Rapid purification and metabolomic profiling of synaptic vesicles from mammalian brain.

Lynne Chantranupong,Wengang Wang,Michael E Pacold,Bernardo L Sabatini,Drew R Jones,Jessica L Saulnier

doi:10.7554/elife.59699

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 SV 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.

Highlights

Critical to the function of the brain are neurotransmitters, a diverse class of small molecules that act as chemical messengers between neurons
Neurotransmitters are stored in synaptic vesicles (SVs), membrane-bound organelles located within presynaptic axon terminals and whose activity-dependent release is essential for proper transmission of information within the brain (Jahn and Südhof, 1994)
SVs isolated with SV-Tag are comparable to those purified via lengthier, traditional differential centrifugation protocols, as assessed by immunoblot (Figure S1E) and electron microscopy (Figure S1F), albeit with a reduction in yield that comes as a tradeoff for the speed of isolation. These analyses indicate we were able to enrich for SVs, they do not provide evidence that the SVs are intact, which is crucial for subsequent metabolite analysis

Summary

Introduction

Critical to the function of the brain are neurotransmitters, a diverse class of small molecules that act as chemical messengers between neurons. Upon electrical excitation of a neuron via action potentials, SVs rapidly fuse with the membrane to release their neurotransmitters, which are detected by transmembrane receptors on a postsynaptic neuron (Südhof, 2013; Traynelis et al, 2014). This binding event can trigger diverse consequences to the postsynaptic neuron, depending on the identity of the neurotransmitter and the receptors to which it binds. The functional contribution of a neuron to a circuit is defined by the neurotransmitters it releases and the postsynaptic cells that it contacts

Methods

Results

Conclusion