Kahng, Jessica A, Xavier, Andre M, Ferro, Austin, Tang, Samantha X, Auguste, Yohan SS, Cheadle, Lucas (October 2024) High-confidence and high-throughput quantification of synapse engulfment by oligodendrocyte precursor cells. Nature Protocols. ISSN 1754-2189 (Public Dataset)
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.
| Item Type: | Paper |
|---|---|
| Subjects: | organs, tissues, organelles, cell types and functions organs, tissues, organelles, cell types and functions > sub-cellular tissues: types and functions organs, tissues, organelles, cell types and functions > sub-cellular tissues: types and functions > synapse |
| CSHL Authors: | |
| Communities: | CSHL labs > Cheadle lab School of Biological Sciences > Publications |
| SWORD Depositor: | CSHL Elements |
| Depositing User: | CSHL Elements |
| Date: | 3 October 2024 |
| Date Deposited: | 04 Oct 2024 19:52 |
| Last Modified: | 10 Dec 2024 14:21 |
| Related URLs: | |
| Dataset ID: |
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| URI: | https://repository.cshl.edu/id/eprint/41695 |
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