Integrated high-confidence and high-throughput approaches for quantifying synapse engulfment by oligodendrocyte precursor cells.

Oligodendrocyte precursor cells (OPCs) sculpt neural circuits through the phagocytic engulfment of synapses during development and in adulthood. However, precise techniques for analyzing synapse engulfment by OPCs are limited. Here, we describe a two-pronged cell biological approach for quantifying synapse engulfment by OPCs which merges low-and high-throughput methodologies. In the first method, an adeno-associated virus encoding a pH-sensitive, fluorescently-tagged synaptic marker is expressed in neurons This construct allows for the differential labeling of presynaptic inputs that are contained outside of and within acidic phagolysosomal compartments. When followed by immunostaining for markers of OPCs and synapses in lightly fixed tissue, this approach enables the quantification of synapses engulfed by around 30-50 OPCs within a given experiment. In the second method, OPCs isolated from dissociated brain tissue are fixed, incubated with fluorescent antibodies against presynaptic proteins, and then analyzed by flow cytometry. This approach enables the quantification of presynaptic material within tens of thousands of OPCs in less than one week. These methods extend beyond the current imaging-based engulfment assays designed to quantify synaptic phagocytosis by brain-resident immune cells, microglia. Through the integration of these methods, the engulfment of synapses by OPCs can be rigorously quantified at both the individual and populational levels. With minor modifications, these approaches can be adapted to study synaptic phagocytosis by numerous glial cell types in the brain.