High-confidence and high-throughput quantification of synapse engulfment by oligodendrocyte precursor cells

Area of Science:

• Neuroscience
• Cell Biology
• Developmental Biology

Background:

• Oligodendrocyte precursor cells (OPCs) play a crucial role in neural circuit development and maintenance through synaptic pruning.
• Accurate quantification of synapse engulfment by OPCs is essential for understanding neural circuit sculpting.
• Existing methods for analyzing synapse engulfment by OPCs lack sufficient accuracy and throughput.

Purpose of the Study:

• To develop and validate novel, high-confidence, and high-throughput methodologies for quantifying synapse engulfment by OPCs.
• To provide researchers with improved tools for studying OPC-mediated synaptic pruning.
• To enable the study of synaptic phagocytosis by OPCs at both individual and population levels.

Main Methods:

• A two-pronged approach combining in vivo imaging and ex vivo flow cytometry.
• In vivo method: Adeno-associated virus (AAV) expressing a pH-sensitive fluorescent synaptic marker in neurons, combined with OPC marker immunostaining.
• Ex vivo method: Isolation and fixation of OPCs, followed by incubation with fluorescent presynaptic protein antibodies and flow cytometry analysis.

Main Results:

• The in vivo method allows quantification of synapse engulfment by 30-50 OPCs per experiment.
• The ex vivo flow cytometry method quantifies presynaptic material in tens of thousands of OPCs in under a week.
• Both methods provide accurate and reliable quantification of synaptic engulfment by OPCs.

Conclusions:

• The developed methodologies offer significant improvements in accuracy and throughput for studying synapse engulfment by OPCs.
• These approaches can be adapted to investigate synaptic phagocytosis by various glial cell types.
• The protocols are suitable for researchers with expertise in confocal microscopy and flow cytometry, facilitating broader application in neuroscience research.