Abstract
Oligodendrocyte precursor cells (OPCs) sculpt neural circuits through the phagocytic engulfment of synapses during development and adulthood. However, existing techniques for analyzing synapse engulfment by OPCs have limited accuracy. Here we describe the quantification of synapse engulfment by OPCs via a two-pronged cell biological approach that combines high-confidence and high-throughput methodologies. Firstly, an adeno-associated virus encoding a pH-sensitive, fluorescently tagged synaptic marker is expressed in neurons in vivo to differentially label presynaptic inputs, depending upon whether they are outside of or within acidic phagolysosomal compartments. When paired with immunostaining for OPC markers in lightly fixed tissue, this approach quantifies the engulfment of synapses by around 30–50 OPCs in each experiment. The second method uses OPCs isolated from dissociated brain tissue that are then fixed, incubated with fluorescent antibodies against presynaptic proteins, and analyzed by flow cytometry, enabling the quantification of presynaptic material within tens of thousands of OPCs in <1 week. The integration of both methods extends the current imaging-based assays, originally designed to quantify synaptic phagocytosis by other brain cells such as microglia and astrocytes, by enabling the quantification of synaptic engulfment by OPCs at individual and populational levels. With minor modifications, these approaches can be adapted to study synaptic phagocytosis by numerous glial cell types in the brain. The protocol is suitable for users with expertise in both confocal microscopy and flow cytometry. The imaging-based and flow cytometry-based protocols require 5 weeks and 2 d to complete, respectively.
Key points
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An adeno-associated virus encoding a pH-sensitive, fluorescently tagged synaptic marker is expressed in neurons in vivo and combined with immunostaining for oligodendrocyte precursor cells (OPCs) in fixed tissue to quantify synaptic engulfment within individual OPCs. The measurements can be integrated with data from the flow cytometry-based quantification of synapses within OPCs pooled from across the mouse brain.
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This protocol extends the limited capabilities in throughput and accuracy of existing imaging-based approaches.
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Data availability
Raw microscopy images (.czi files), processed microscopy images (.ims files), and flow cytometry data (.fcs files) have all been deposited and are available in the following Zenodo repository (https://doi.org/10.5281/zenodo.10625432).
Code availability
All R code used for analysis of flow cytometry data can be accessed from our Zenodo repository (https://doi.org/10.5281/zenodo.10625432).
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Acknowledgements
We acknowledge and thank the following individuals for their contributions: U. Vrudhula for contributions to early imaging-based engulfment protocols; A.-S. Nichitiu for previously validating the pSynDig construct; P. Moody from the CSHL Flow Cytometry Core; E. Wee from the CSHL Microscopy Core and M.J. Gastinger from Imaris/Andor. This work was supported by the following funding sources (to L.C.): R00MH120051, DP2MH132943, R01NS131486, Rita Allen Scholar Award, McKnight Scholar Award, Klingenstein-Simons Fellowship Award in Neuroscience, Pershing Square Innovation Fund and a Brain and Behavior Foundation NARSAD grant. L.C. is a Howard Hughes Medical Institute Freeman Hrabowski Scholar.
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J.A.K., A.M.X. and L.C. wrote the paper. For the material, reagents and protocol sections, the components describing the microscopy-based approach were written by J.A.K. and the components related to flow cytometry were written by A.M.X. A.F. designed and produced the pSynDig construct. Y.S.S.A. designed the analysis of pSynDig data. A.F., J.A.K., S.T. and Y.A. contributed to optimizing the pSynDig OPC engulfment assay analysis. All figures were created by J.A.K., A.M.X. and L.C. Supervision and funding provided by L.C.
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Nature Protocols thanks Tara DeSilva, Xiaoping Tong and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Auguste, Y. S. S. et al. Nat. Neurosci. 25, 1273–1278 (2022): https://doi.org/10.1038/s41593-022-01170-x
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Kahng, J.A., Xavier, A.M., Ferro, A. et al. High-confidence and high-throughput quantification of synapse engulfment by oligodendrocyte precursor cells. Nat Protoc (2024). https://doi.org/10.1038/s41596-024-01048-1
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DOI: https://doi.org/10.1038/s41596-024-01048-1
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