Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

A rapid Percoll gradient procedure for preparation of synaptosomes

Abstract

Homogenization of fresh brain tissue in isotonic medium shears plasma membranes causing nerve terminals to become separated from their axons and postsynaptic connections. The nerve terminal membranes then reseal to form synaptosomes. The discontinuous Percoll gradient procedure described here is designed to isolate synaptosomes from brain homogenates in the minimum time to allow functional experiments to be performed. Synaptosomes are isolated using a medium-speed centrifuge, while maintaining isotonic conditions and minimizing mechanically damaging resuspension steps. This protocol has advantages over other procedures in terms of speed and by producing relatively homogeneous synaptosomes, minimizing the presence of synaptic and glial plasma membranes and extrasynaptosomal mitochondria. The purified synaptosomes are viable and take up and release neurotransmitters very efficiently. A typical yield of synaptosomes is between 2.5 and 4 mg of synaptosomal protein per gram rat brain. The procedure takes 1 h from homogenization of the brain until collection of the synaptosomal suspension from the Percoll gradient.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: The Percoll gradient procedure.
Figure 2: Electron microscopy of Percoll gradient fractions.
Figure 3: Generating Percoll discontinuous step gradients with a peristaltic pump (Step 9A).
Figure 4: Generating Percoll discontinuous step gradients manually with a pipettor (Step 9B).

Similar content being viewed by others

References

  1. Whittaker, V.P. & Gray, E.G. The synapse: biology and morphology. Br. Med. Bull. 18, 223–228 (1962).

    Article  CAS  PubMed  Google Scholar 

  2. Dunkley, P.R. et al. The preparation and use of synaptosomes for studying secretion of catecholamines. In The Secretory Process Vol. 3 309–328 (Eds. A. Poisner and J.M. Tritaro. Elsevier, Amsterdam, 1987).

    Google Scholar 

  3. Thorne, B., Sanderson, E.M., Wonnacott, S. & Dunkley, P.R. Isolation of hippocampal synaptosomes on Percoll gradients: cholinergic and noradrenergic markers. J. Neurochem. 56, 479–484 (1991).

    Article  CAS  PubMed  Google Scholar 

  4. Whittaker, V.P. Thirty years of synaptosome research. J. Neurocytol. 22, 735–742 (1993).

    Article  CAS  PubMed  Google Scholar 

  5. Gray, E.G. & Whittaker, V.P. The isolation of nerve endings from brain: an electron-microscopic study of cell fragments derived by homogenization and centrifugation. J. Anat. 96, 79–88 (1962).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. De Robertis, E., Pellegrino de Iraldi, A., Rodriquez de Lores Arniaz, G. & Salganicoff, L. Cholinergic and noncholinergic nerve endings in rat brain. I. Isolation and subcellular distribution of acetylcholine and acetylcholinesterase. J. Neurochem. 9, 23–35 (1962).

    Article  CAS  PubMed  Google Scholar 

  7. Dunkley, P.R., Jarvie, P.E. & Rostas, J.A. Distribution of calmodulin- and cyclic AMP-stimulated protein kinases in synaptosomes. J. Neurochem. 51, 57–68 (1988).

    Article  CAS  PubMed  Google Scholar 

  8. Whittaker, V.P., Michaelson, I.A. & Kirkland, R.J. The separation of synaptic vesicles from nerve-ending particles ('synaptosomes'). Biochem. J. 90, 293–303 (1964).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Polosa, P.L. & Attardi, G. Distinctive pattern and translational control of mitochondrial protein synthesis in rat brain synaptic endings. J. Biol. Chem. 266, 10011–10017 (1991).

    CAS  PubMed  Google Scholar 

  10. Dunkley, P.R. & Robinson, P.J. Depolarisation-dependent protein phosphorylation in synaptosomes: mechanisms and significance. Prog. Brain Res. 69, 273–293 (1986).

    Article  CAS  PubMed  Google Scholar 

  11. Ashton, A.C. & Ushkaryov, Y.A. Properties of synaptic vesicle pools in mature central nerve terminals. J. Biol. Chem. 280, 37278–37288 (2005).

    Article  CAS  PubMed  Google Scholar 

  12. Morgan, I.G. Synaptosomes and cell separation. Neuroscience 1, 159–165 (1976).

    Article  CAS  PubMed  Google Scholar 

  13. Balázs, R. et al. Subcellular fractionation of rat cerebellum: an electron microscopic and biochemical investigation. III. Isolation of large fragments of the cerebellar glomeruli. Brain Res. 86, 17–30 (1975).

    Article  PubMed  Google Scholar 

  14. Neal, M.J. & Atterwill, C.K. Isolation of photoreceptor and conventional nerve terminals by subcellular fractionation of rabbit retina. Nature 251, 331–333 (1971).

    Article  Google Scholar 

  15. Wolf, M.E. & Kapatos, G. Flow cytometric analysis and isolation of permeabilized dopamine nerve terminals from rat striatum. J. Neurosci. 9, 106–114 (1989).

    Article  CAS  PubMed  Google Scholar 

  16. De Belleroche, J., Dykes, C.R. & Thomas, A.J. The automated separation and analysis of dopamine, its amino acid precursors and metabolites, and the application of the method to the measurement of specific radioactivities of dopamine in striatial synaptosomes. Anal. Biochem. 71, 193–203 (1976).

    Article  CAS  PubMed  Google Scholar 

  17. De Belleroche, J.S. & Bradford, H.F. On the site of origin of transmitter amino acids released by depolarization of nerve terminals in vitro . J. Neurochem. 29, 335–343 (1977).

    Article  CAS  PubMed  Google Scholar 

  18. Dodd, P.R. et al. A rapid method for preparing synaptosomes: comparison, with alternative procedures. Brain Res. 226, 107–118 (1981).

    Article  CAS  PubMed  Google Scholar 

  19. Abdel-Latif, A.A. A simple method for isolation of nerve-ending particles from rat brain. Biochim. Biophys. Acta 121, 403–406 (1966).

    Article  CAS  PubMed  Google Scholar 

  20. Cotman, C.W. & Matthews, D.A. Synaptic plasma membranes from rat brain synaptosomes: isolation and partial characterization. Biochim. Biophys. Acta 249, 380–394 (1971).

    Article  CAS  PubMed  Google Scholar 

  21. Booth, R.F. & Clark, J.B. A rapid method for the preparation of relatively pure metabolically competent synaptosomes from rat brain. Biochem. J. 176, 365–370 (1978).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Enriquez, J.A., Sanchez-Prieto, J., Muino Blanco, M.T., Hernandez-Yago, J. & Lopez-Perez, M.J. Rat brain synaptosomes prepared by phase partition. J. Neurochem. 55, 1841–1849 (1990).

    Article  CAS  PubMed  Google Scholar 

  23. Nichols, R.A., Wu, W.C., Haycock, J.W. & Greengard, P. Introduction of impermeant molecules into synaptosomes using freeze/thaw permeabilization. J. Neurochem. 52, 521–529 (1989).

    Article  CAS  PubMed  Google Scholar 

  24. Nichols, R.A., Chilcote, T.J., Czernik, A.J. & Greengard, P. Synapsin I regulates glutamate release from rat brain synaptosomes. J. Neurochem. 58, 783–785 (1992).

    Article  CAS  PubMed  Google Scholar 

  25. Gleitz, J., Beile, A., Wilffert, B. & Tegtmeier, F. Cryopreservation of freshly isolated synaptosomes prepared from the cerebral cortex of rats. J. Neurosci. Methods 47, 191–197 (1993).

    Article  CAS  PubMed  Google Scholar 

  26. Hardy, J.A. et al. Metabolically active synaptosomes can be prepared from frozen rat and human brain. J. Neurochem. 40, 608–614 (1983).

    Article  CAS  PubMed  Google Scholar 

  27. Eshleman, A.J., Wolfrum, K., Mash, D.C., Christensen, K. & Janowsky, A. Drug interactions with the dopamine transporter in cryopreserved human caudate. J. Pharmacol. Exp. Ther. 296, 442–449 (2001).

    CAS  PubMed  Google Scholar 

  28. Nagy, A.K., Houser, C.R. & Delgado-Escueta, A.V. Synaptosomal ATPase activities in temporal cortex and hippocampal formation of humans with focal epilepsy. Brain Res. 529, 192–201 (1990).

    Article  CAS  PubMed  Google Scholar 

  29. Sherman, A.D., Hegwood, T.S., Baruah, S. & Waziri, R. Deficient NMDA-mediated glutamate release from synaptosomes of schizophrenics. Biol. Psychiatry 30, 1191–1198 (1991).

    Article  CAS  PubMed  Google Scholar 

  30. Heinonen, E. & Akerman, K.E. Measurement of cytoplasmic, free magnesium concentration with entrapped eriochrome blue in nerve endings isolated from the guinea pig brain. Neurosci. Lett. 72, 105–110 (1986).

    Article  CAS  PubMed  Google Scholar 

  31. Raiteri, M., Sala, R., Fassio, A., Rossetto, O. & Bonanno, G. Entrapping of impermeant probes of different size into nonpermeabilized synaptosomes as a method to study presynaptic mechanisms. J. Neurochem. 74, 423–431 (2000).

    Article  CAS  PubMed  Google Scholar 

  32. Onofri, F. et al. Synapsin phosphorylation by SRC tyrosine kinase enhances SRC activity in synaptic vesicles. J. Biol. Chem. 282, 15754–15767 (2007).

    Article  CAS  PubMed  Google Scholar 

  33. Hens, J.J. et al. N-terminal-specific anti-B-50 (GAP-43) antibodies inhibit Ca(2+)-induced noradrenaline release, B-50 phosphorylation and dephosphorylation, and calmodulin binding. J. Neurochem. 64, 1127–1136 (1995).

    Article  CAS  PubMed  Google Scholar 

  34. Pertoft, H., Rubin, K., Kjellén, L., Laurent, T.C. & Klingeborn, B. The viability of cells grown or centrifuged in a new density gradient medium, Percoll(TM). Exp. Cell Res. 110, 449–457 (1977).

    Article  CAS  PubMed  Google Scholar 

  35. Pertoft, H., Laurent, T.C., Laas, T. & Kagedal, L. Density gradients prepared from colloidal silica particles coated by polyvinylpyrrolidone (Percoll). Anal. Biochem. 88, 271–282 (1978).

    Article  CAS  PubMed  Google Scholar 

  36. Nagy, A. & Delgado-Escueta, A.V. Rapid preparation of synaptosomes from mammalian brain using nontoxic isoosmotic gradient material (Percoll). J. Neurochem. 43, 1114–1123 (1984).

    Article  CAS  PubMed  Google Scholar 

  37. Dunkley, P.R., Jarvie, P.E., Heath, J.W., Kidd, G.J. & Rostas, J.A.P. A rapid method for isolation of synaptosomes on percoll gradients. Brain Res. 372, 115–129 (1986).

    Article  CAS  PubMed  Google Scholar 

  38. Dunkley, P.R. et al. A rapid Percoll gradient procedure for isolation of synaptosomes directly from an S1 fraction: homogeneity and morphology of subcellular fractions. Brain Res. 441, 59–71 (1988).

    Article  CAS  PubMed  Google Scholar 

  39. Harrison, S.M., Jarvie, P.E. & Dunkley, P.R. A rapid Percoll gradient procedure for isolation of synaptosomes directly from an S1 fraction: viability of subcellular fractions. Brain Res. 441, 72–80 (1988).

    Article  CAS  PubMed  Google Scholar 

  40. Cousin, M.A. & Robinson, P.J. Two mechanisms of synaptic vesicle recycling in rat brain nerve terminals. J. Neurochem. 75, 1645–1653 (2000).

    Article  CAS  PubMed  Google Scholar 

  41. Thorne, B., Irons, J., Lunt, G.G., Wonnacott, S. & Dunkley, P.R. Comparison of methods for rapid isolation of synaptosomes from brain regions for uptake and release studies. Biochem. Soc. Trans. 16, 309–310 (1989).

    Article  Google Scholar 

  42. Ghijsen, W.E., Leenders, A.G. & Lopes da Silva, F.H. Regulation of vesicle traffic and neurotransmitter release in isolated nerve terminals. Neurochem. Res. 28, 1443–1452 (2003).

    Article  CAS  PubMed  Google Scholar 

  43. Raiteri, L. & Raiteri, M. Synaptosomes still viable after 25 years of superfusion. Neurochem. Res. 25, 1265–1274 (2000).

    Article  CAS  PubMed  Google Scholar 

  44. Nicholls, D.G. Bioenergetics and transmitter release in the isolated nerve terminal. Neurochem. Res. 28, 1433–1441 (2003).

    Article  CAS  PubMed  Google Scholar 

  45. Shetty, P.K., Huang, F.L. & Huang, K.P. Ischemia-elicited oxidative modulation of Ca2+/calmodulin-dependent protein kinase II. J. Biol. Chem. 283, 5389–5401 (2008).

    Article  CAS  PubMed  Google Scholar 

  46. Mokin, M., Zheng, Z. & Keifer, J. Conversion of silent synapses into the active pool by selective GluR1-3 and GluR4 AMPAR trafficking during in vitro classical conditioning. J. Neurophysiol. 98, 1278–1286 (2007).

    Article  CAS  PubMed  Google Scholar 

  47. Verhage, M., Besselsen, E., Lopes da Silva, F.H. & Ghijsen, W.E. Ca2+-dependent regulation of presynaptic stimulus-secretion coupling. J. Neurochem. 53, 1188–1194 (1989).

    Article  CAS  PubMed  Google Scholar 

  48. Robinson, P.J. & Lovenberg, W. Dopamine and serotonin in two populations of synaptosomes isolated by percoll gradient centrifugation. Neurochem. Int. 9, 455–458 (1986).

    Article  CAS  PubMed  Google Scholar 

  49. Robinson, P.J., Gehlert, D.R., Sanna, E. & Hanbauer, I. Two fractions enriched for striatal synaptosomes isolated by percoll gradient centrifugation: synaptosome morphology, dopamine and serotonin receptor distribution, and adenylate cyclase activity. Neurochem. Int. 15, 339–348 (1989).

    Article  CAS  PubMed  Google Scholar 

  50. Taupin, P., Zini, S., Cesselin, F., Ben-Ari, Y. & Roisin, M.P. Subcellular fractionation on Percoll gradient of mossy fiber synaptosomes: morphological and biochemical characterization in control and degranulated rat hippocampus. J. Neurochem. 62, 1586–1595 (1994).

    Article  CAS  PubMed  Google Scholar 

  51. Martin, H., Rostas, J., Patel, Y. & Aitken, A. Subcellular localisation of 14-3-3 isoforms in rat brain using specific antibodies. J. Neurochem. 63, 2259–2265 (1994).

    Article  CAS  PubMed  Google Scholar 

  52. Sherman, A.D. Isolation of metabolically distinct synaptosomes on Percoll gradients. Neurochem. Res. 14, 97–101 (1989).

    Article  CAS  PubMed  Google Scholar 

  53. Asermely, K.E. & O'Neill, J.J. Vesamicol, an inhibitor of acetylcholine vesicle packaging, increases synaptophysin phosphorylation in rat cortical synaptosomes. Life Sci. 59, 2113–2128 (1996).

    Article  CAS  PubMed  Google Scholar 

  54. Faúndez, V., Krauss, R., Holuigue, L., Garrido, J. & Gonzalez, A. Epidermal growth factor receptor in synaptic fractions of the rat central nervous system. J. Biol. Chem. 267, 20363–20370 (1992).

    PubMed  Google Scholar 

  55. Matsumoto, I., Combs, M.R. & Jones, D.J. Characterization of 5-hydroxytryptamine1B receptors in rat spinal cord via [125I]iodocyanopindolol binding and inhibition of [3H]-5-hydroxytryptamine release. J. Pharmacol. Exp. Ther. 260, 614–626 (1992).

    CAS  PubMed  Google Scholar 

  56. Sims, N.R. Rapid isolation of metabolically active mitochondria from rat brain and subregions using Percoll density gradient centrifugation. J. Neurochem. 55, 698–707 (1990).

    Article  CAS  PubMed  Google Scholar 

  57. Sims, N.R. & Anderson, M.F. Isolation of mitochondria from rat brain using Percoll density gradient centrifugation. Nat. Protoc. 3, 1228–1239 (2008).

    Article  CAS  PubMed  Google Scholar 

  58. Brown, M.R., Sullivan, P.G. & Geddes, J.W. Synaptic mitochondria are more susceptible to Ca2+overload than nonsynaptic mitochondria. J. Biol. Chem. 281, 11658–11668 (2006).

    Article  CAS  PubMed  Google Scholar 

  59. Nakamura, Y., Iga, K., Shibata, T., Shudo, M. & Kataoka, K. Glial plasmalemmal vesicles: a subcellular fraction from rat hippocampal homogenate distinct from synaptosomes. Glia 9, 48–56 (1993).

    Article  CAS  PubMed  Google Scholar 

  60. Stigliani, S. et al. Glia re-sealed particles freshly prepared from adult rat brain are competent for exocytotic release of glutamate. J. Neurochem. 96, 656–668 (2006).

    Article  CAS  PubMed  Google Scholar 

  61. Westphalen, R.I. & Hemmings, H.C. Jr. Volatile anesthetic effects on glutamate versus GABA release from isolated rat cortical nerve terminals: basal release. J. Pharmacol. Exp. Ther. 316, 208–215 (2006).

    Article  CAS  PubMed  Google Scholar 

  62. Westphalen, R.I. & Hemmings, H.C. Jr. Volatile anesthetic effects on glutamate versus GABA release from isolated rat cortical nerve terminals: 4-aminopyridine-evoked release. J. Pharmacol. Exp. Ther. 316, 216–223 (2006).

    Article  CAS  PubMed  Google Scholar 

  63. Lowry, O.H., Rosebrough, N.J., Farr, A.L. & Randall, R.J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275 (1951).

    CAS  Google Scholar 

Download references

Acknowledgements

We thank Professors J. Rostas and S. Wonnacott, Associate Professor J. Heath, Dr G. Kidd, Ms P. Glenfield and Mr S. Harrison for their contributions at different times to the development of the method presented here and Dr L. Bak for the photographs in Figures 1, 3 and 4. This work was supported by the National Health and Medical Research Council of Australia.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Peter R Dunkley.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Dunkley, P., Jarvie, P. & Robinson, P. A rapid Percoll gradient procedure for preparation of synaptosomes. Nat Protoc 3, 1718–1728 (2008). https://doi.org/10.1038/nprot.2008.171

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2008.171

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing