Live imaging of synapse engulfment by microglia in vitro

[Speaker] Megumi Andoh:1
[Co-author] Ryuta Koyama:1, Yuji Ikegaya:1
1:Laboratory of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Japan

Synaptic pruning is fundamental for the refinement of synaptic connections. Accumulating reports have suggested that the brain-resident immune cells microglia are the executioner of synaptic pruning. Major skepticism to this idea is based on the fact that no report has successfully captured an actual moment of synaptic pruning by microglia both in vivo and in vitro. Here, we investigated whether and how microglia prune synapses from living neurons, using a live imaging system for neuron-glia cocultures. Firstly, we prepared the general culture system of dispersed microglia on glass dishes but failed to reproduce the ramified shape of microglia that is observed in vivo. Because the morphology of microglia is relevant to their function, we needed a proper in vitro system in which the ramified shape of microglia is maintained. For this purpose, we cocultured microglia with astrocytes and neurons so that the surrounding milieu was similar to in vivo conditions. Under these experimental conditions, microglia now exhibited ramified-like morphology. To time-lapse image these microglia, the glial cocultures were prepared from CX3CR1GFP/+ mice whose microglia express GFP. Neurons were transfected with synaptophysin-RFP after added to the glial cocultures. Using this coculture system, we observed a translocation of RFP-labeled synaptophysin from the axons of living neurons to GFP-labeled microglial processes. We are now investigating whether neuronal activity regulates synaptic pruning by pharmacological modulations. Our experimental system is expected to clarify the processes and mechanisms of synaptic pruning by microglia.
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