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Small-scale isolation of synaptic vesicles from mammalian brain

Nature Protocols volume 8, pages 998–1009 (2013)Cite this article

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Abstract

Synaptic vesicles (SVs) are essential organelles that participate in the release of neurotransmitters from a neuron. Biochemical analysis of purified SVs was instrumental in the identification of proteins involved in exocytotic membrane fusion and neurotransmitter uptake. Although numerous protocols have been published detailing the isolation of SVs from the brain, those that give the highest-purity vesicles often have low yields. Here we describe a protocol for the small-scale isolation of SVs from mouse and rat brain. The procedure relies on standard fractionation techniques, including differential centrifugation, rate-zonal centrifugation and size-exclusion chromatography, but it has been optimized for minimal vesicle loss while maintaining a high degree of purity. The protocol can be completed in less than 1 d and allows the recovery of ∼150 μg of vesicle protein from a single mouse brain, thus allowing vesicle isolation from transgenic mice.

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Figure 1
Figure 2: Schematic of an improved procedure for small-scale isolation of SVs from mammalian brain.
Figure 3: Effects of size-exclusion chromatography on SV purity.
Figure 4: Separation by SDS-PAGE of subfractions taken during the isolation of SVs.
Figure 5: Testing SV function.

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Acknowledgements

We thank M. Druminski for excellent technical assistance during the project. E. Reisinger and P. D'Adamo provided synaptobrevin 1-GFP knock-in mice and GDI knockout mice, respectively, which were used in 'proof-of-principle' experiments during protocol development. I. Herford prepared rat hippocampal cultures. G. van den Bogaart and H. Martens (Synaptic Systems) gave much useful advice. S.A. is a student of the Gauss PhD program of the Georg-August University, Göttingen. This work was funded by the Max Planck Society (R.J.) and by a European Research Council Starting Grant (AstroFunc: 281961) (M.H.).

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Author notes

  1. Saheeb Ahmed and Matthew Holt: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany

    Saheeb Ahmed, Matthew Holt & Reinhard Jahn

  2. Laboratory of Glia Biology, Vlaams Instituut voor Biotechnologie (VIB) Center for the Biology of Disease, Katholieke Universiteit (KU) Leuven, Leuven, Belgium

    Matthew Holt

  3. Electron Microscopy Group, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany

    Dietmar Riedel

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  1. Saheeb Ahmed
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Contributions

The experimental work was performed and analyzed by S.A. and M.H., with the exception of the electron microscopy, which was performed by D.R. The project was conceived and supervised by R.J., in conjunction with M.H. S.A., M.H. and R.J. wrote the manuscript.

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Correspondence to Reinhard Jahn.

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The authors declare no competing financial interests.

Supplementary information

Images of pellets and supernatants obtained during SV purification.

Flow diagram of synaptic vesicle purification, illustrating pellets and supernatants formed at each stage. Careful handling of pellets and supernatants is essential to avoid contamination in the final synaptic vesicle fraction. When removing the S1 supernatant, do not disturb any of the white material surrounding the P1 pellet (red arrowhead). Likewise, when resuspending the P2 (synaptosomes) avoid the brown material in the middle of the pellet (red arrowhead), which is enriched in mitochondria. After centrifugation of the sucrose cushion, proteasome contamination will be enriched on the top of the sucrose layer and should be avoided (red boxed region in both low magnification (left) and high magnification (right) images), while a vesicle rich pellet will be formed at the bottom of the tube (red circles). (PDF 1938 kb)

Optimisation of the sucrose cushion step.

Representative immunoblots of fractions taken from the sucrose cushion, following centrifugation under different conditions. Fractions were tested for the presence of synaptophysin (synaptic vesicle marker) or Rpt4 (proteasome marker); in our experience, proteasomes are the major contaminant of the synaptic vesicle preparation. Different relative volumes of the input fraction (CS1) to 0.7M sucrose cushion were used. Centrifugation forces and times for a 70.1 Ti rotor were also systematically varied (although only data for 5ml CS1 on a 5ml cushion centrifuged at 65,000 rpm (400,000 gmax; condition 1), 54,000 rpm (270,000 gmax; condition 2) and 38,000 rpm (133,000 gmax; condition 3) are shown). Note the differential separation of Rpt4 from synaptophysin under the various conditions. Best separation was found using a 0.7M sucrose cushion centrifuged at 38,000 rpm for 1h. (PDF 1137 kb)

Monitoring vesicle purity by immunoblotting of subcellular fractions.

Subfractions taken during the isolation of synaptic vesicles were separated by SDSPAGE and immunoblotted to determine the distribution profiles of various marker proteins (additional to those shown in Figure 4B). Some markers ERC1b/2 (active zone protein), PSD-95 (post-synaptic scaffolding protein) and Rab-GDI (regulator of Rab protein activity) should be absent from the purified vesicle fraction. The remaining protein profiles are for known residents of the plasma membrane (syntaxin 1A and SNAP-25, or for known interacting partners, such as Munc-18). While the degree of plasma membrane contamination within the synaptic vesicle fraction is known to be low (as judged by Na+/K+-ATPase immunoreactivity), it is conceivable that some plasma membrane proteins may use synaptic vesicles as part of their recycling pathway. This is especially true for syntaxin 1 and SNAP-25, that are both members of the synaptic core-complex essential for vesicle fusion, and which may be recycled to some degree with synaptic vesicles. (PDF 406 kb)

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Ahmed, S., Holt, M., Riedel, D. et al. Small-scale isolation of synaptic vesicles from mammalian brain. Nat Protoc 8, 998–1009 (2013). https://doi.org/10.1038/nprot.2013.053

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