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Isolation and Characterization of Sympathetic Extracellular Vesicles

Research on Extracellular Vesicles (EVs) is increasing at a rapid pace; therefore, it is crucial to maintain rigor in characterizing EVs from a new model system. Neuronal derived EVs have been well described in the central nervous system; however, studies in the peripheral nervous system have largely focused on EVs derived from supporting cell types such as endothelial cells or glia. Additionally, EVs are heterogeneous in size, shape, cargo and biogenic origin and therefore a multimodal approach to characterization must be used. Here we conduct a thorough description of EVs derived from sympathetic neurons using immunoblot assays, nanoparticle tracking analysis and cryo-electron microscopy. We show that primary sympathetic cultures secrete EVs in a density-dependent manner and that their sizing aligns with those reported in the literature. Lastly, using a compartmentalized culture system we show that EVs secreted by the somatodendritic domain of neurons contain cargo that originated at their distal axon. This work establishes foundational protocols to explore the biogenesis and function of EVs in the peripheral nervous system.