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
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
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
Similar content being viewed by others
References
Whittaker, V.P. & Gray, E.G. The synapse: biology and morphology. Br. Med. Bull. 18, 223–228 (1962).
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).
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).
Whittaker, V.P. Thirty years of synaptosome research. J. Neurocytol. 22, 735–742 (1993).
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).
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).
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).
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).
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).
Dunkley, P.R. & Robinson, P.J. Depolarisation-dependent protein phosphorylation in synaptosomes: mechanisms and significance. Prog. Brain Res. 69, 273–293 (1986).
Ashton, A.C. & Ushkaryov, Y.A. Properties of synaptic vesicle pools in mature central nerve terminals. J. Biol. Chem. 280, 37278–37288 (2005).
Morgan, I.G. Synaptosomes and cell separation. Neuroscience 1, 159–165 (1976).
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).
Neal, M.J. & Atterwill, C.K. Isolation of photoreceptor and conventional nerve terminals by subcellular fractionation of rabbit retina. Nature 251, 331–333 (1971).
Wolf, M.E. & Kapatos, G. Flow cytometric analysis and isolation of permeabilized dopamine nerve terminals from rat striatum. J. Neurosci. 9, 106–114 (1989).
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).
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).
Dodd, P.R. et al. A rapid method for preparing synaptosomes: comparison, with alternative procedures. Brain Res. 226, 107–118 (1981).
Abdel-Latif, A.A. A simple method for isolation of nerve-ending particles from rat brain. Biochim. Biophys. Acta 121, 403–406 (1966).
Cotman, C.W. & Matthews, D.A. Synaptic plasma membranes from rat brain synaptosomes: isolation and partial characterization. Biochim. Biophys. Acta 249, 380–394 (1971).
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).
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).
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).
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).
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).
Hardy, J.A. et al. Metabolically active synaptosomes can be prepared from frozen rat and human brain. J. Neurochem. 40, 608–614 (1983).
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).
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).
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).
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).
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).
Onofri, F. et al. Synapsin phosphorylation by SRC tyrosine kinase enhances SRC activity in synaptic vesicles. J. Biol. Chem. 282, 15754–15767 (2007).
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).
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).
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).
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).
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).
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).
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).
Cousin, M.A. & Robinson, P.J. Two mechanisms of synaptic vesicle recycling in rat brain nerve terminals. J. Neurochem. 75, 1645–1653 (2000).
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).
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).
Raiteri, L. & Raiteri, M. Synaptosomes still viable after 25 years of superfusion. Neurochem. Res. 25, 1265–1274 (2000).
Nicholls, D.G. Bioenergetics and transmitter release in the isolated nerve terminal. Neurochem. Res. 28, 1433–1441 (2003).
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).
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).
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).
Robinson, P.J. & Lovenberg, W. Dopamine and serotonin in two populations of synaptosomes isolated by percoll gradient centrifugation. Neurochem. Int. 9, 455–458 (1986).
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).
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).
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).
Sherman, A.D. Isolation of metabolically distinct synaptosomes on Percoll gradients. Neurochem. Res. 14, 97–101 (1989).
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).
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).
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).
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).
Sims, N.R. & Anderson, M.F. Isolation of mitochondria from rat brain using Percoll density gradient centrifugation. Nat. Protoc. 3, 1228–1239 (2008).
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).
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).
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).
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).
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).
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).
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
Corresponding author
Rights 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
Published:
Issue Date:
DOI: https://doi.org/10.1038/nprot.2008.171
This article is cited by
-
Neuronal MHC-I complex is destabilized by amyloid-β and its implications in Alzheimer’s disease
Cell & Bioscience (2023)
-
Enhanced presynaptic mitochondrial energy production is required for memory formation
Scientific Reports (2023)
-
Modification of astrocytic Cx43 hemichannel activity in animal models of AD: modulation by adenosine A2A receptors
Cellular and Molecular Life Sciences (2023)
-
Beta-2 adrenergic receptor agonism alters astrocyte phagocytic activity and has potential applications to psychiatric disease
Discover Mental Health (2023)
-
Fear extinction is regulated by the activity of long noncoding RNAs at the synapse
Nature Communications (2023)
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.