Rapid purification and metabolomic profiling of synaptic vesicles from mammalian brain
- Harvard Medical School, United States
- NYU Langone Health, United States
- Howard Hughes Medical Institute, Harvard Medical School, United States
Abstract
Neurons communicate by the activity-dependent release of small-molecule neurotransmitters packaged into synaptic vesicles (SVs). Although many molecules have been identified as neurotransmitters, technical limitations have precluded a full metabolomic analysis of synaptic vesicle content. Here, we present a workflow to rapidly isolate SVs and to interrogate their metabolic contents at high-resolution using mass spectrometry. We validated the enrichment of glutamate in SVs of primary cortical neurons using targeted polar metabolomics. Unbiased and extensive global profiling of SVs isolated from these neurons revealed that the only detectable polar metabolites they contain are the established neurotransmitters glutamate and GABA. In addition, we adapted the approach to enable quick capture of SVs directly from brain tissue and determined the neurotransmitter profiles of diverse brain regions in a cell-type specific manner. The speed, robustness, and precision of this method to interrogate SV contents will facilitate novel insights into the chemical basis of neurotransmission.
Data availability
All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for all figures.
Article and author information
Author details
Bernardo L Sabatini
Funding
Howard Hughes Medical Institute (Investigator)
- Bernardo L Sabatini
National Institute of Neurological Disorders and Stroke (R37NS046579)
- Bernardo L Sabatini
Howard Hughes Medical Institute (Hanna Gray Fellowship)
- Lynne Chantranupong
Mary Kay Foundation (Cancer Research Grant 017-032)
- Michael E Pacold
Hearst Foundation (V Foundation V Scholar Grant (V2017-004))
- Michael E Pacold
National Cancer Institute (K22 Career Transition Award (1K22CA212059))
- Michael E Pacold
NIH (R01 NS108151-01)
- Drew R Jones
FNIH (RFA 2018-PACT001)
- Drew R Jones
NIH (HHS-NIH-NIAD-BAA2018)
- Drew R Jones
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: All experimental manipulations were performed in accordance with protocols (#03551) approved by the Harvard Standing Committee on Animal Care following guidelines described in the US National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Copyright
© 2020, Chantranupong et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Rapid purification and metabolomic profiling of synaptic vesicles from mammalian brain
eLife 9:e59699.
https://doi.org/10.7554/eLife.59699
