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Subcellular Fractionation of Brain Tissue from Small Tissue Explants

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Book cover Synaptosomes

Part of the book series: Neuromethods ((NM,volume 141))

Abstract

For several decades, neurobiologists have used subcellular fractionation methods to analyze the molecular structure and some functional features of the cells in the central nervous system. Indeed, the brain tissue is built through the networking of neuronal, glial, and vascular cells in an intermingled meshwork of micrometer-sized structures. Subcellular fractionation protocols allow for the separation of specific compartments such as synapses (called “synaptosomes”), synaptic plasma membranes, and synaptic vesicles for their analysis at the molecular level. Most current protocols were established to process large amounts of tissue as required in previous experimental paradigms. Here, we provide a protocol to prepare synaptosomes from as little as 10 mg of tissue or a full fractionation to enrich crude synaptic vesicles and synaptic plasma membranes from 20 mg of tissue. This protocol will be useful to anyone aiming at addressing specific questions regarding local microcircuits in combination with connectomics and proteomics approaches.

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References

  1. Brody TM, Bain JA (1951) Effect of barbiturates on oxidative phosphorylation. Proc Soc Exp Biol Med 77:50–53

    Article  CAS  Google Scholar 

  2. Brody TM, Bain JA (1952) A mitochondrial preparation from brain. Biol Chem 195:685

    CAS  Google Scholar 

  3. Spanner S (1972) Methods of separating the subcellular components of brain tissue. In: Zambotti V, Tettamanti G, Arrigoni M (eds) Glycolipids, glycoproteins, and mucopolysaccharides of the nervous system. Advances in experimental medicine and biology, vol 25. Plenum Press, New York, pp 195–207

    Chapter  Google Scholar 

  4. Whitakker VP (1959) The isolation and characterization of acetylcholine-containing particles from brain. Biochem J 72:694–706

    Article  Google Scholar 

  5. Whittaker VP, Michaelson IA, Kirkland RJ (1964) The separation of synaptic vesicles from nerve-ending particles (‘synaptosomes’). Biochem J 90:293–303

    Article  CAS  Google Scholar 

  6. Whittaker VP (1965) The application of subcellular fractionation techniques to the study of brain function. Prog Biophys Mol Biol 15:39–96

    Article  CAS  Google Scholar 

  7. Whittaker VP (1993) Thirty years of synaptosome research. J Neurocytol 22:735–742

    Article  CAS  Google Scholar 

  8. Gray EG, Whitakker VP (1962) The isolation of nerve endings from brain: an electron-microscopic study of cell fragments derived by homogenization and centrifugation. J Anat 96:79–88

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Zimmermann H, Fonnum F (2016) Victor P. Whittaker (1919-2016). J Neurochem 139:333–335. https://doi.org/10.1111/jnc.13778

    Article  CAS  PubMed  Google Scholar 

  10. Huttner WB, Schiebler W, Greengard P, De Camilli P (1983) Synapsin I (protein I), a nerve terminal-specific phosphoprotein. III. Its association with synaptic vesicles studied in a highly purified synaptic vesicle preparation. J Cell Biol 96:1374–1388

    Article  CAS  Google Scholar 

  11. Cotman CW, Matthews DA (1971) Synaptic plasma membranes from rat brain synaptosomes: isolation and partial characterization. Biochim Biophys Acta 249:380–394

    Article  CAS  Google Scholar 

  12. Jones DH, Matus AI (1974) Isolation of synaptic plasma membrane from brain by combined flotation-sedimentation density gradient centrifugation. Biochim Biophys Acta 356:276–287

    Article  CAS  Google Scholar 

  13. Boyken J, Grønborg M, Riedel D, Urlaub H, Jahn R, Chua JJ (2013) Molecular profiling of synaptic vesicle docking sites reveals novel proteins but few differences between glutamatergic and GABAergic synapses. Neuron 78:285–297. https://doi.org/10.1016/j.neuron.2013.02.027

    Article  CAS  PubMed  Google Scholar 

  14. Weingarten J, Lassek M, Mueller BF, Rohmer M, Lunger I, Baeumlisberger D, Dudek S, Gogesch P, Karas M, Volknandt W (2014) The proteome of the presynaptic active zone from mouse brain. Mol Cell Neurosci 59:106–118. https://doi.org/10.1016/j.mcn.2014.02.003

    Article  CAS  PubMed  Google Scholar 

  15. Luquet E, Biesemann C, Munier A, Herzog E (2017) Purification of synaptosome populations using fluorescence-activated synaptosome sorting. Methods Mol Biol 1538:121–134

    Article  CAS  Google Scholar 

  16. De Robertis E, Rodriguez De Lores Arnaiz G, Pellegrino De Iraldi A (1962) Isolation of synaptic vesicles from nerve endings of the rat brain. Nature 194:794–795

    Article  Google Scholar 

  17. Bajjalieh SM, Frantz GD, Weimann JM, McConnell SK, Scheller RH (1994) Differential expression of synaptic vesicle protein 2 (SV2) isoforms. J Neurosci 14:5223–5235

    Article  CAS  Google Scholar 

  18. Burre J, Beckhaus T, Schagger H, Corvey C, Hofmann S, Karas M, Zimmermann H, Volknandt W (2006) Analysis of the synaptic vesicle proteome using three gel-based protein separation techniques. Proteomics 6:6250–6262. https://doi.org/10.1002/pmic.200600357

    Article  CAS  PubMed  Google Scholar 

  19. Farr CD, Gafken PR, Norbeck AD, Doneanu CE, Stapels MD, Barofsky DF, Minami M, Saugstad JA (2004) Proteomic analysis of native metabotropic glutamate receptor 5 protein complexes reveals novel molecular constituents. J Neurochem 91:438–450. https://doi.org/10.1111/j.1471-4159.2004.02735.x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Gronborg M, Pavlos NJ, Brunk I, Chua JJ, Munster-Wandowski A, Riedel D, Ahnert-Hilger G, Urlaub H, Jahn R (2010) Quantitative comparison of glutamatergic and GABAergic synaptic vesicles unveils selectivity for few proteins including MAL2, a novel synaptic vesicle protein. J Neurosci 30:2–12. https://doi.org/10.1523/JNEUROSCI.4074-09.2010

    Article  CAS  PubMed  Google Scholar 

  21. Husi H, Ward MA, Choudhary JS, Blackstock WP, Grant SG (2000) Proteomic analysis of NMDA receptor-adhesion protein signalling complexes. Nat Neurosci 3:661–669

    Article  CAS  Google Scholar 

  22. Lassek M, Weingarten J, Einsfelder U, Brendel P, Muller U, Volknandt W (2013) Amyloid precursor proteins are constituents of the presynaptic active zone. J Neurochem 127:48–56. https://doi.org/10.1111/jnc.12358

    Article  CAS  PubMed  Google Scholar 

  23. Lassek M, Weingarten J, Volknandt W (2015) The synaptic proteome. Cell Tissue Res 359:255–265. https://doi.org/10.1007/s00441-014-1943-4

    Article  CAS  PubMed  Google Scholar 

  24. Morciano M, Burre J, Corvey C, Karas M, Zimmermann H, Volknandt W (2005) Immunoisolation of two synaptic vesicle pools from synaptosomes: a proteomics analysis. J Neurochem 95:1732–1745. https://doi.org/10.1111/j.1471-4159.2005.03506.x

    Article  CAS  PubMed  Google Scholar 

  25. Morciano M, Beckhaus T, Karas M, Zimmermann H, Volknandt W (2009) The proteome of the presynaptic active zone: from docked synaptic vesicles to adhesion molecules and maxi-channels. J Neurochem 108:662–675. https://doi.org/10.1111/j.1471-4159.2008.05824.x

    Article  CAS  PubMed  Google Scholar 

  26. Weingarten J, Lassek M, Mueller BF et al (2014) The proteome of the presynaptic active zone from mouse brain. Mol Cell Neurosci 59C:106–118. https://doi.org/10.1016/j.mcn.2014.02.003

    Article  CAS  Google Scholar 

  27. Yao J, Nowack A, Kensel-Hammes P, Gardner RG, Bajjalieh SM (2010) Cotrafficking of SV2 and synaptotagmin at the synapse. J Neurosci 30:5569–5578. https://doi.org/10.1523/JNEUROSCI.4781-09.2010

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Biesemann C, GronborgM LE et al (2014) Proteomic screening of glutamatergic mouse brain synaptosomes isolated by fluorescence activated sorting. EMBO J 33:157–170. https://doi.org/10.1002/embj.201386120

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Tannu NS, Hemby SE (2006) Methods for proteomics in neuroscience. Prog Brain Res 158:41–82. https://doi.org/10.1016/S0079-6123(06)58003-3

    Article  CAS  PubMed  Google Scholar 

  30. Schreiner D, Savas JN, Herzog E, Brose N, de Wit J (2017) Synapse biology in the ‘circuit-age’-paths toward molecular connectomics. Curr Opin Neurobiol 42:102–110. https://doi.org/10.1016/j.conb.2016.12.004

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

MFA salary was supported by the Fondation Pour la Recherche Médicale (ING20150532192), and EH received the following funding from the French Agence Nationale de la Recherche: ANR-10-LABX-43 BRAIN and ANR-12-JSV4-0005-01 VGLUT-IQ. PT received the following funding: ANR-10-IDEX-03-02, NARSAD Young investigator grant from the brain and behavior foundation, Région Aquitaine, INRA. Experiments were performed thanks to the equipment of the Bordeaux Neurocampus central facility for biochemistry and biophysics of protein.

Author information

Author notes

  1. Etienne Herzog and Maria Florencia Angelo are contributed equally to this work.

Authors and Affiliations

  1. INRA, Nutrition et Neurobiologie Intégrée, UMR 1286, Bordeaux, France

    Véronique De-Smedt-Peyrusse & Pierre Trifilieff

  2. Univ. Bordeaux, Nutrition et Neurobiologie Intégrée, UMR 1286, Bordeaux, France

    Véronique De-Smedt-Peyrusse & Pierre Trifilieff

  3. Univ. Bordeaux, IINS, UMR 5297, Bordeaux, France

    Laetitia Darriet, Etienne Herzog & Maria Florencia Angelo

  4. CNRS, IINS, UMR 5297, Bordeaux, France

    Laetitia Darriet, Etienne Herzog & Maria Florencia Angelo

  5. Lycée Fernand Daguin, Académie de Bordeaux, Mérignac, France

    Laetitia Darriet

Authors
  1. Véronique De-Smedt-Peyrusse
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  2. Laetitia Darriet
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  3. Pierre Trifilieff
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  4. Etienne Herzog
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  5. Maria Florencia Angelo
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Corresponding author

Correspondence to Maria Florencia Angelo .

Editor information

Editors and Affiliations

  1. Department of Psychology, Neuroscience & Behavior, McMaster University, Hamilton, ON, Canada

    Kathryn M. Murphy

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De-Smedt-Peyrusse, V., Darriet, L., Trifilieff, P., Herzog, E., Angelo, M.F. (2018). Subcellular Fractionation of Brain Tissue from Small Tissue Explants. In: Murphy, K. (eds) Synaptosomes. Neuromethods, vol 141. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8739-9_5

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  • DOI: https://doi.org/10.1007/978-1-4939-8739-9_5

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8738-2

  • Online ISBN: 978-1-4939-8739-9

  • eBook Packages: Springer Protocols

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