Live Imaging and Characterization of Microglia Dynamics and Interactions with Synapses in Diseased Murine Retina

Microglia are the resident macrophages of the central nervous system (CNS) that respond to tissue infection and injury. In addition to their role in inflammation, microglia play a developmental role in circuit refinement through synaptic pruning. However, the mechanisms of synaptic pruning in neuroinflammation and neurodegeneration remain unknown. In this protocol, we use a mouse retina explant model to study microglia dynamics ex vivo. To examine microglia motility and their interactions with postsynaptic proteins, we label synapses with AAV-PSD95-RFP and record timelapse videos of motile microglia colocalized with postsynaptic proteins using spinning disk confocal microscopy. We then create surface and spot reconstructions of microglia and PSD95 using image analysis software. Data such as microglia displacement length, process speed, and contact with postsynaptic puncta can then be extracted from these surfaces to understand microglia behavior both in homeostatic states and after neuronal injury. This protocol can be useful in examining the role of microglia in synaptic pruning in retinal neurodegenerative diseases.