High-confidence and high-throughput quantification of synapse engulfment by oligodendrocyte precursor cells.
Journal
Nature protocols
ISSN: 1750-2799
Titre abrégé: Nat Protoc
Pays: England
ID NLM: 101284307
Informations de publication
Date de publication:
03 Oct 2024
03 Oct 2024
Historique:
received:
06
05
2023
accepted:
10
07
2024
medline:
4
10
2024
pubmed:
4
10
2024
entrez:
3
10
2024
Statut:
aheadofprint
Résumé
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.
Identifiants
pubmed: 39363108
doi: 10.1038/s41596-024-01048-1
pii: 10.1038/s41596-024-01048-1
doi:
Types de publication
Journal Article
Review
Langues
eng
Sous-ensembles de citation
IM
Subventions
Organisme : U.S. Department of Health & Human Services | NIH | National Institute of Mental Health (NIMH)
ID : R00MH120051
Organisme : U.S. Department of Health & Human Services | NIH | National Institute of Mental Health (NIMH)
ID : DP2MH132943
Organisme : U.S. Department of Health & Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)
ID : R01NS131486
Organisme : Rita Allen Foundation (RAF)
ID : Rita Allen Scholar
Organisme : McKnight Foundation
ID : McKnight Scholar
Organisme : Esther A. and Joseph Klingenstein Fund (Esther A. & Joseph Klingenstein Fund, Inc.)
ID : Klingenstein-Simons Fellowship in Neuroscience
Organisme : Brain and Behavior Research Foundation (Brain & Behavior Research Foundation)
ID : NARSAD grant
Informations de copyright
© 2024. Springer Nature Limited.
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