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Synaptosome Preparations: Which Procedure Should I Use?

  • Peter R. Dunkley
  • Phillip J. Robinson
Protocol
Part of the Neuromethods book series (NM, volume 141)

Abstract

One of the most extensively used model systems to investigate the functions and the chemical control of the nerve terminal has been the synaptosome. Synaptosomes are pinched-off nerve endings that form by shearing forces during homogenization of neuronal tissue. Depending on the aim of the study, synaptosomes can be used once they are formed within the whole neuronal tissue homogenate, or they can be separated from other subcellular organelles and enriched to various extents, depending on the fractionation procedures adopted. Each procedure varies in the time that it takes and provides synaptosomes with different levels of homogeneity and viability. The major contaminants of synaptosomes that remain after fractionation include neuronal and glial plasma membranes, attached postsynaptic membranes and densities, microsomes, synaptic vesicles, and extra-synaptosomal mitochondria. This chapter documents the most commonly used procedures for making synaptosomes, indicates the most likely contaminants present after each procedure, and assesses the viability of the resulting synaptosomes. This provides researchers with a decision tool to determine which synaptosome preparation procedure best suits the aims of their study.

Key words

Synaptosomes Formation Fractionation Homogeneity Viability 

Notes

Acknowledgments

We would like to thank all of our colleagues who have helped to develop the Percoll gradient procedure for preparation of synaptosomes [9, 11, 26, 30, 67] for their hard work, camaraderie, and intellectual input. The NHMRC of Australia is thanked for past funding of projects arising from the use of synaptosomes.

References

  1. 1.
    Del Castillo J, Katz B (1956) Biophysical aspects of neuro-muscular transmission. Prog Biophys Biophys Chem 6:121–170CrossRefGoogle Scholar
  2. 2.
    Hebb CO, Whittaker VP (1958) Intracellular distributions of acetylcholine and choline acetylase. J Physiol 142(1):187–196PubMedPubMedCentralCrossRefGoogle Scholar
  3. 3.
    Derobertis E et al (1962) Cholinergic and non-cholinergic nerve endings in rat brain .1. Isolation and subcellular distribution of acetylcholine and acetylcholinesterase. J Neurochem 9:23–35CrossRefGoogle Scholar
  4. 4.
    Gray EG, Whittaker VP (1962) Isolation of nerve endings from brain - an electron-microscopic study of cell fragments derived by homogenization and centrifugation. J Anat 96:79–88PubMedPubMedCentralGoogle Scholar
  5. 5.
    Whittaker VP, Gray EG (1962) Synapse - biology and morphology. Br Med Bull 18(3):223–228CrossRefGoogle Scholar
  6. 6.
    Whittaker VP, Michaelson IA, Kirkland RJ (1964) Separation of synaptic vesicles from nerve-ending particles (synaptosomes ). Biochem J 90(2):293–303PubMedPubMedCentralCrossRefGoogle Scholar
  7. 7.
    Rockland KS (2002) Non-uniformity of extrinsic connections and columnar organization. J Neurocytol 31(3–5):247–253PubMedCrossRefGoogle Scholar
  8. 8.
    Faisal AA, White JA, Laughlin SB (2005) Ion-channel noise places limits on the miniaturization of the brain’s wiring. Curr Biol 15(12):1143–1149PubMedCrossRefGoogle Scholar
  9. 9.
    Dunkley PR et al (1986) A rapid method for isolation of synaptosomes on Percoll gradients. Brain Res 372(1):115–129CrossRefGoogle Scholar
  10. 10.
    Robinson PJ, Lovenberg W (1986) Dopamine and serotonin in two populations of synaptosomes isolated by percoll gradient centrifugation. Neurochem Int 9(3):455–458CrossRefGoogle Scholar
  11. 11.
    Dunkley PR et al (1988) A rapid Percoll gradient procedure for isolation of synaptosomes directly from an S1 fraction: homogeneity and morphology of subcellular fractions. Brain Res 441(1–2):59–71CrossRefGoogle Scholar
  12. 12.
    Wilhelm BG et al (2014) Composition of isolated synaptic boutons reveals the amounts of vesicle trafficking proteins. Science 344(6187):1023–1028PubMedCrossRefGoogle Scholar
  13. 13.
    Hollingsworth EB et al (1985) Biochemical characterization of a filtered synaptoneurosome preparation from Guinea pig cerebral cortex: cyclic adenosine 3′:5′-monophosphate-generating systems, receptors, and enzymes. J Neurosci 5(8):2240–2253CrossRefGoogle Scholar
  14. 14.
    Schwartz RD et al (1984) Barbiturate and picrotoxin-sensitive chloride efflux in rat cerebral cortical synaptoneurosomes. FEBS Lett 175(1):193–196PubMedCrossRefGoogle Scholar
  15. 15.
    Whittaker VP, Greengard P (1971) Isolation of synaptosomes from brain of a teleost fish, centriopristes-striatus. J Neurochem 18(2):173–176Google Scholar
  16. 16.
    Babitch JA et al (1976) Preparation of chick brain synaptosomes and synaptosomal membranes. Biochim Biophys Acta 433(1):75–89PubMedCrossRefGoogle Scholar
  17. 17.
    Morgan IG et al (1971) Isolation of plasma membranes from rat brain. Biochim Biophys Acta 241(3):737–751PubMedCrossRefGoogle Scholar
  18. 18.
    Gurd JW et al (1974) Isolation and partial characterization of rat brain synaptic plasma membranes. J Neurochem 22(2):281–290PubMedCrossRefGoogle Scholar
  19. 19.
    Cotman C, Mahler HR, Anderson NG (1968) Isolation of a membrane fraction enriched in nerve-end membranes from rat brain by zonal cetrifugation. Biochim Biophys Acta 163(2):272–275PubMedCrossRefGoogle Scholar
  20. 20.
    Leskawa KC et al (1979) Large-scale preparation of synaptosomes from bovine brain using a zonal rotor technique. Neurochem Res 4(4):483–504PubMedCrossRefGoogle Scholar
  21. 21.
    Kishi M et al (1991) Pharmacological characteristics of choline transport system in mouse cerebral cortical neurons in primary culture. Jpn J Pharmacol 55(2):223–232PubMedCrossRefGoogle Scholar
  22. 22.
    Restituito S et al (2011) Synaptic autoregulation by metalloproteases and gamma-secretase. J Neurosci 31(34):12083–12093PubMedPubMedCentralCrossRefGoogle Scholar
  23. 23.
    Bate C, Williams A (2012) Neurodegeneration induced by clustering of sialylated glycosylphosphatidylinositols of prion proteins. J Biol Chem 287(11):7935–7944PubMedPubMedCentralCrossRefGoogle Scholar
  24. 24.
    Leshchyns’ka I et al (2015) Abeta-dependent reduction of NCAM2-mediated synaptic adhesion contributes to synapse loss in Alzheimer’s disease. Nat Commun 6:8836PubMedPubMedCentralCrossRefGoogle Scholar
  25. 25.
    Faundez V et al (1992) Epidermal growth factor receptor in synaptic fractions of the rat central nervous system. J Biol Chem 267(28):20363–20370PubMedGoogle Scholar
  26. 26.
    Thorne B, Wonnacott S, Dunkley PR (1991) Isolation of hippocampal synaptosomes on Percoll gradients: cholinergic markers and ligand binding sites. J Neurochem 56(2):479–484CrossRefGoogle Scholar
  27. 27.
    De Belleroche JA, Bradford HF (1975) The release of endogenous 3,4-dihydroxyphenethylamine from synaptosomes isolated from corpus striatum. Biochem Soc Trans 3(1):99–101CrossRefGoogle Scholar
  28. 28.
    Clark M, Dar MS (1989) Release of endogenous glutamate from rat cerebellar synaptosomes: interactions with adenosine and ethanol. Life Sci 44(22):1625–1635CrossRefGoogle Scholar
  29. 29.
    Tamir H, Rapport MM, Roizin L (1974) Preparation of synaptosomes and vesicles with sodium diatrizoate. J Neurochem 23(5):943–949CrossRefGoogle Scholar
  30. 30.
    Dunkley PR, Jarvie PE, Robinson PJ (2008) A rapid Percoll gradient procedure for preparation of synaptosomes. Nat Protoc 3(11):1718–1728CrossRefGoogle Scholar
  31. 31.
    Hardy JA et al (1983) Metabolically active synaptosomes can be prepared from frozen rat and human brain. J Neurochem 40(3):608–614PubMedCrossRefGoogle Scholar
  32. 32.
    Dodd PR et al (1986) Optimization of freezing, storage, and thawing conditions for the preparation of metabolically active synaptosomes from frozen rat and human brain. Neurochem Pathol 4(3):177–198CrossRefGoogle Scholar
  33. 33.
    Dodd PR et al (1989) Uptake of gamma-aminobutyric acid and L-glutamic acid by synaptosomes from postmortem human cerebral cortex: multiple sites, sodium dependence and effect of tissue preparation. Brain Res 490(2):320–331CrossRefGoogle Scholar
  34. 34.
    Mazzo F et al (2016) Reconstitution of synaptic ion channels from rodent and human brain in Xenopus oocytes: a biochemical and electrophysiological characterization. J Neurochem 138(3):384–396PubMedPubMedCentralCrossRefGoogle Scholar
  35. 35.
    Franklin W, Taglialatela G (2016) A method to determine insulin responsiveness in synaptosomes isolated from frozen brain tissue. J Neurosci Methods 261:128–134PubMedPubMedCentralCrossRefGoogle Scholar
  36. 36.
    Wilson WS, Cooper JR (1972) Preparation of cholinergic synaptosomes from bovine superior cervical ganglia. J Neurochem 19(12):2779–2790PubMedCrossRefGoogle Scholar
  37. 37.
    Dowdall MJ, Whittaker VP (1973) Comparative studies in synaptosome formation: preparation of synaptosomes from head ganglion of squid, Loligo-Pealii. J Neurochem 20(4):921–935Google Scholar
  38. 38.
    Newkirk RF et al (1976) Comparative studies in synaptosome formation: preparation of synaptosomes from the ventral nerve cord of the lobster (Homarus americanus). Brain Res 101(1):103–111PubMedCrossRefGoogle Scholar
  39. 39.
    Israel M et al (1985) Large-scale purification of Torpedo electric organ synaptosomes. J Neurochem 44(4):1107–1110PubMedCrossRefGoogle Scholar
  40. 40.
    Chin GJ et al (1989) Aplysia synaptosomes. I. Preparation and biochemical and morphological characterization of subcellular membrane fractions. J Neurosci 9(1):38–48PubMedCrossRefGoogle Scholar
  41. 41.
    Bisby MA, Fillenz M (1969) Isolation of peripheral synaptosomes from a sympathetically innervated tissue. J Physiol 204(2):105P+PubMedGoogle Scholar
  42. 42.
    Bisby MA, Fillenz M (1971) The storage of endogenous noradrenaline in sympathetic nerve terminals. J Physiol 215(1):163–179PubMedPubMedCentralCrossRefGoogle Scholar
  43. 43.
    Simon EJ et al (1976) Comparative studies on synaptosomes - applicability of rapid method for preparing synaptosomes to elasmobranch brain. Neurochem Res 1(1):83–92PubMedCrossRefGoogle Scholar
  44. 44.
    Lagercrantz H, Pertoft H (1972) Separation of catecholamine storing synaptosomes in colloidal silica density gradients. J Neurochem 19(3):811–823PubMedCrossRefGoogle Scholar
  45. 45.
    Chin GJ, Shapiro E, Schwartz JH (1989) Aplysia synaptosomes. II. Release of transmitters. J Neurosci 9(1):49–55PubMedCrossRefGoogle Scholar
  46. 46.
    Hebb CO, Smallman BN (1956) Intracellular distribution of choline acetylase. J Physiol 134(2):385–392PubMedPubMedCentralCrossRefGoogle Scholar
  47. 47.
    Abdel-Latif AA (1966) A simple method for isolation of nerve-ending particles from rat brain. Biochim Biophys Acta 121(2):403–406PubMedPubMedCentralCrossRefGoogle Scholar
  48. 48.
    Kurokawa M, Sakamoto T, Kato M (1965) A rapid isolation of nerve-ending particles from brain. Biochim Biophys Acta 94(1):307–309CrossRefGoogle Scholar
  49. 49.
    Ashton AC, Ushkaryov YA (2005) Properties of synaptic vesicle pools in mature central nerve terminals. J Biol Chem 280(44):37278–37288CrossRefGoogle Scholar
  50. 50.
    Nagy A, Delgado-Escueta AV (1984) Rapid preparation of synaptosomes from mammalian brain using nontoxic isoosmotic gradient material (Percoll). J Neurochem 43(4):1114–1123CrossRefGoogle Scholar
  51. 51.
    Geddes JW, Newstead JD, Wood JD (1980) Stability of the glutamate content of synaptosomes during their preparation. Neurochem Res 5(10):1107–1116PubMedCrossRefGoogle Scholar
  52. 52.
    Whittaker VP (1959) The isolation and characterization of acetylcholine-containing particles from brain. Biochem J 72:694–706PubMedPubMedCentralCrossRefGoogle Scholar
  53. 53.
    Dodd PR et al (1981) Rapid preparation of nerve ending particles (synaptosomes) from rat-brain - comparison with 2 standard methods. J Anat 132:462Google Scholar
  54. 54.
    Cotman C et al (1970) Analytical differential centrifugation: an analysis of the sedimentation properties of synaptosomes, mitochondria and lysosomes from rat brain homogenates. Arch Biochem Biophys 136(2):436–447PubMedCrossRefGoogle Scholar
  55. 55.
    Cotman CW, Matthews DA (1971) Synaptic plasma membranes from rat brain synaptosomes: isolation and partial characterization. Biochim Biophys Acta 249(2):380–394PubMedPubMedCentralCrossRefGoogle Scholar
  56. 56.
    Hajos F (1975) An improved method for the preparation of synaptosomal fractions in high purity. Brain Res 93(3):485–489PubMedCrossRefGoogle Scholar
  57. 57.
    Cotman C, Herschman H, Taylor D (1971) Subcellular fractionation of cultured glial cells. J Neurobiol 2(2):169–180PubMedCrossRefGoogle Scholar
  58. 58.
    Whittaker VP (1968) The morphology of fractions of rat forebrain synaptosomes separated on continuous sucrose density gradients. Biochem J 106(2):412–417PubMedPubMedCentralCrossRefGoogle Scholar
  59. 59.
    Van der Krogt JA, Koot-Gronsveld E, Van den Berg CJ (1983) Subcellular fractionation of striatum: sedimentation properties of dopaminergic synaptosomes. Life Sci 33(7):605–613PubMedCrossRefGoogle Scholar
  60. 60.
    Kornguth SE, Anderson JW, Scott G (1969) Isolation of synaptic complexes in a caesium chloride density gradient: electron microscopic and immunohistochemical studies. J Neurochem 16(3):1017–1024PubMedCrossRefGoogle Scholar
  61. 61.
    Autilio LA et al (1968) Biochemical studies of synapses in vitro. I. Protein synthesis. Biochemistry 7(7):2615–2622PubMedCrossRefGoogle Scholar
  62. 62.
    Day ED et al (1971) Zonal centrifuge profiles of rat brain homogenates - instability in sucrose, stability in Iso-Osomotic Ficoll-sucrose. Anal Biochem 39(1):29–45PubMedCrossRefGoogle Scholar
  63. 63.
    Booth RF, Clark JB (1978) A rapid method for the preparation of relatively pure metabolically competent synaptosomes from rat brain. Biochem J 176(2):365–370PubMedPubMedCentralCrossRefGoogle Scholar
  64. 64.
    Verity MA (1972) Cation modulation of synaptosomal respiration. J Neurochem 19(5):1305–1317CrossRefGoogle Scholar
  65. 65.
    Jansen GJ, Vaneerten MTW, Forrester IT (1982) Biochemical events associated with Ficoll washing of ram spermatozoa. Proc N Z Soc Anim Prod 42:95–97Google Scholar
  66. 66.
    Asgeirsson D et al (2006) Increased glomerular permeability to negatively charged Ficoll relative to neutral Ficoll in rats. Am J Physiol Renal Physiol 291(5):F1083–F1089PubMedCrossRefGoogle Scholar
  67. 67.
    Harrison SM, Jarvie PE, Dunkley PR (1988) A rapid Percoll gradient procedure for isolation of synaptosomes directly from an S1 fraction: viability of subcellular fractions. Brain Res 441(1–2):72–80PubMedCrossRefGoogle Scholar
  68. 68.
    Docherty M, Bradford HF, Wu JY (1987) The preparation of highly purified GABAergic and cholinergic synaptosomes from mammalian brain. Neurosci Lett 81(1–2):232–238PubMedCrossRefGoogle Scholar
  69. 69.
    Bowman D, Smith W, Mccormack A (1995) Affinity purification of rat cortical and chicken forebrain synaptosomes using a biotinylated derivative of omega-Cgtx Gvia. Neuropharmacology 34(7):743–752PubMedCrossRefGoogle Scholar
  70. 70.
    Bowman D et al (1994) Affinity purification of synaptosomes using a biotinylated derivative of Omega-Conotoxin Gvia. Br J Pharmacol 112:U176–U176CrossRefGoogle Scholar
  71. 71.
    Enriquez JA et al (1990) Rat brain synaptosomes prepared by phase partition. J Neurochem 55(6):1841–1849PubMedCrossRefGoogle Scholar
  72. 72.
    Muino-Blanco T et al (1993) Use of a resolving density gradient created with dextran and poly(ethylene glycol) to purify brain synaptosomes. J Biochem Biophys Methods 27(1):1–10PubMedCrossRefGoogle Scholar
  73. 73.
    Whittaker VP (1993) Thirty years of synaptosome research. J Neurocytol 22(9):735–742PubMedPubMedCentralCrossRefGoogle Scholar
  74. 74.
    Robinson PJ et al (1994) Phosphorylation of dynamin-I and synaptic-vesicle recycling. Trends Neurosci 17(8):348–353PubMedCrossRefGoogle Scholar
  75. 75.
    Wolf ME, Kapatos G (1989) Flow cytometric analysis of rat striatal nerve terminals. J Neurosci 9(1):94–105PubMedCrossRefGoogle Scholar
  76. 76.
    Gylys KH, Fein JA, Cole GM (2000) Quantitative characterization of crude synaptosomal fraction (P-2) components by flow cytometry. J Neurosci Res 61(2):186–192PubMedPubMedCentralCrossRefGoogle Scholar
  77. 77.
    Wolf ME, Zigmond MJ, Kapatos G (1989) Tyrosine hydroxylase content of residual striatal dopamine nerve terminals following 6-hydroxydopamine administration: a flow cytometric study. J Neurochem 53(3):879–885PubMedCrossRefGoogle Scholar
  78. 78.
    Sokolow S et al (2012) Isolation of synaptic terminals from Alzheimer’s disease cortex. Cytometry A 81(3):248–254CrossRefGoogle Scholar
  79. 79.
    Wang DS et al (2005) Decreased neprilysin immunoreactivity in Alzheimer disease, but not in pathological aging. J Neuropathol Exp Neurol 64(5):378–385PubMedCrossRefGoogle Scholar
  80. 80.
    Sokolow S et al (2015) Pre-synaptic C-terminal truncated tau is released from cortical synapses in Alzheimer’s disease. J Neurochem 133(3):368–379PubMedPubMedCentralCrossRefGoogle Scholar
  81. 81.
    Gylys KH, Bilousova T (2017) Flow cytometry analysis and quantitative characterization of tau in synaptosomes from Alzheimer’s disease brains. Methods Mol Biol 1523:273–284PubMedPubMedCentralCrossRefGoogle Scholar
  82. 82.
    Postupna NO et al (2014) Flow cytometry analysis of synaptosomes from post-mortem human brain reveals changes specific to Lewy body and Alzheimer’s disease. Lab Investig 94(10):1161–1172PubMedPubMedCentralCrossRefGoogle Scholar
  83. 83.
    Biesemann C et al (2014) Proteomic screening of glutamatergic mouse brain synaptosomes isolated by fluorescence activated sorting. EMBO J 33(2):157–170PubMedPubMedCentralCrossRefGoogle Scholar
  84. 84.
    Luquet E et al (2017) Purification of synaptosome populations using fluorescence-activated synaptosome sorting. Methods Mol Biol 1538:121–134CrossRefGoogle Scholar
  85. 85.
    Daniel JA et al (2012) Analysis of synaptic vesicle endocytosis in synaptosomes by high-content screening. Nat Protoc 7(8):1439–1455CrossRefGoogle Scholar
  86. 86.
    Choi SW, Gerencser AA, Nicholls DG (2009) Bioenergetic analysis of isolated cerebrocortical nerve terminals on a microgram scale: spare respiratory capacity and stochastic mitochondrial failure. J Neurochem 109(4):1179–1191PubMedPubMedCentralCrossRefGoogle Scholar
  87. 87.
    Marcelli S et al (2016) Targeting SUMO-1ylation contrasts synaptic dysfunction in a mouse model of Alzheimer’s disease. Mol Neurobiol 54(8):6609–6623PubMedCrossRefGoogle Scholar
  88. 88.
    Chi P, Greengard P, Ryan TA (2003) Synaptic vesicle mobilization is regulated by distinct synapsin I phosphorylation pathways at different frequencies. Neuron 38(1):69–78PubMedCrossRefGoogle Scholar
  89. 89.
    Darcy KJ et al (2006) Constitutive sharing of recycling synaptic vesicles between presynaptic boutons. Nat Neurosci 9(3):315–321PubMedCrossRefGoogle Scholar

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Authors and Affiliations

  1. 1.School of Biomedical Sciences and Pharmacy, Hunter Medical Research InstituteThe University of NewcastleCallaghanAustralia
  2. 2.Cell Signalling Unit, Children’s Medical Research InstituteThe University of SydneyWentworthvilleAustralia

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