Isolation of Synaptic Vesicles

Branch Craige1, Gloria Salazar1, Victor Faundez1

1 Emory University, Atlanta, Georgia
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 3.12
DOI:  10.1002/0471143030.cb0312s25
Online Posting Date:  December, 2004
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Abstract

Synaptic vesicles are the most abundant secretory organelle in eukaryotic neural cells. Synaptic vesicles are physically distinct from other membrane‐bound organelles because they are small, spherical, and highly uniform in size with a diameter of about 40 nm. Synaptic vesicles also have a characteristically low density because water and phospholipids account for 88% of an individual synaptic vesicle's weight. The homogeneous size and density of the synaptic vesicle are characteristics that are exploited in most synaptic vesicle isolation procedures. Synaptic vesicles are purified by isopycnic/velocity sedimentation and size‐based purification schemes. However, protocols differ in the tissue source of vesicles, the way the tissue is homogenized, and the way the vesicles are fractionated. This unit describes two well‐characterized and widely used synaptic vesicle isolation procedures that are capable of generating membrane fractions that are 20 to 30 times enriched in synaptic vesicles.

     
 
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Table of Contents

  • Basic Protocol 1: Clift‐O'Grady Method of Synaptic Vesicle Isolation
  • Alternate Protocol 1: Huttner Method of Synaptic Vesicle Isolation
  • Synaptic Vesicle Enrichment
  • Basic Protocol 2: Synaptic Vesicle Enrichment by Glycerol Velocity Sedimentation
  • Alternate Protocol 2: Synaptic Vesicle Enrichment by Controlled Pore Glass (CPG) Chromatography
  • Alternate Protocol 3: Synaptic Vesicle Enrichment by Gel‐Filtration Chromatography in Sephacryl S‐1000
  • Support Protocol 1: Synaptic Vesicle Sedimentation at High Speed
  • Support Protocol 2: Immunopurification of SV Subpopulations
  • Support Protocol 3: Tube Siliconization
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Clift‐O'Grady Method of Synaptic Vesicle Isolation

  Materials
  • 200‐ to 250‐g female rats (22 animals needed)
  • Buffer A (see recipe) prepared in H 2O (light water), with and without 1× protease inhibitors
  • 1000 × protease inhibitors (see recipe) or Boehringer Complete protease inhibitor tablets (Roche Applied Science)
  • Buffer A (see recipe) prepared in 80% (v/v) H 2O/20% D 2O (deuterium oxide, 99% isotopic purity), with and without 1× protease inhibitors
  • 320 mM sucrose/4 mM HEPES, pH 7.4, prepared with 100% D 2O
  • 320 mM sucrose/4 mM HEPES, pH 7.4, prepared with 85% (v/v) D 2O/15% H 2O
  • 320 mM sucrose/4 mM HEPES, pH 7.4, prepared with 70% (v/v) D 2O/30% H 2O
  • Dissecting instruments
  • Waring Blendor with small glass cup (kept in cold room)
  • Refrigerated centrifuge with Sorvall SS‐34 rotor (or equivalent), and corresponding polycarbonate centrifuge tubes
  • Beckman ultracentrifuge with 45 Ti rotor (or equivalent), and corresponding Oak Ridge ultracentrifuge tubes
  • Small Teflon‐coated spatula
  • Tight‐clearance Teflon/glass Potter‐Elvehjem homogenizer, ∼50 ml
  • Beckman ultracentrifuge with SW 41 Ti rotor (or equivalent) and corresponding polyallomer ultracentrifuge tubes
  • Loose‐clearance Teflon/glass Potter‐Elvehjem homogenizer, ∼2 ml
  • SG series gradient maker (Hoefer Scientific)
  • Loose‐clearance Teflon/glass Potter‐Elvehjem homogenizer, ∼1 ml
NOTE: Carry out all procedures at 4°C using solutions prechilled to that temperature.

Alternate Protocol 1: Huttner Method of Synaptic Vesicle Isolation

  • 200‐ to 250‐g female rats (14 animals needed)
  • SB buffers (see recipe), with and without 1× protease inhibitors
  • 1 M HEPES buffer, pH 7.4
  • Loose clearance Potter‐Elvehjem (Teflon/glass) homogenizer, ∼50 ml
  • 10‐ml syringe with 25‐G needle
  • Beckman ultracentrifuge with SW 28 rotor (or equivalent), and corresponding Ultraclear centrifuge tubes
NOTE: Carry out all procedures at 4°C using solutions prechilled to that temperature.

Basic Protocol 2: Synaptic Vesicle Enrichment by Glycerol Velocity Sedimentation

  Materials
  • 5% and 25% (v/v) glycerol in buffer A (see recipe for buffer)
  • Synaptic vesicle preparation (see protocol 1 or protocol 2)
  • SG series gradient maker (Hoefer Scientific)
  • Transi‐stir motor (Talboys Engineering Corp.)
  • Beckman ultracentrifuge with SW 55 Ti rotor (or equivalent) and corresponding polyallomer centrifuge tubes
  • Tube‐piercing apparatus (Brandel)

Alternate Protocol 2: Synaptic Vesicle Enrichment by Controlled Pore Glass (CPG) Chromatography

  Materials
  • Glycerol‐coated controlled pore glass (glyceryl‐CPG) beads, 3000 Å particle size (CPG, Inc.)
  • 300 mM glycine/5 mM HEPES, pH 7.4, containing 1× protease inhibitors (see recipe for protease inhibitors)
  • Synaptic vesicle preparation (see protocol 1 or protocol 2)
  • Buffer A (see recipe) containing 1× protease inhibitors (see recipe)
  • 85 × 1.6–cm glass chromatography column, siliconized (see protocol 8)
  • Wall‐mounted column rack
  • Peristaltic pump and fraction collector, with appropriate tubing and connections
  • Additional reagents and equipment for protein assay ( appendix 3B or appendix 3H) and concentration of protein solutions ( appendix 3C)

Alternate Protocol 3: Synaptic Vesicle Enrichment by Gel‐Filtration Chromatography in Sephacryl S‐1000

  Materials
  • Sephacryl S‐1000 resin (Amersham Biosciences)
  • Buffer A (see recipe) containing 25% (v/v) glycerol and 1× protease inhibitors or 300 mM glycine/5 mM HEPES, pH 7.4, containing 1× protease inhibitors (see recipe for protease in hibitors)
  • Synaptic vesicle preparation (see protocol 1 or protocol 2)
  • 15 × 1–cm plastic chromatography column, siliconized (see protocol 8)
  • Additional reagents and equipment for concentrating protein solutions ( appendix 3C)

Support Protocol 1: Synaptic Vesicle Sedimentation at High Speed

  Materials
  • Synaptic vesicle preparation (see previous protocols)
  • Buffer A (see recipe)
  • 1000 × protease inhibitors (see recipe) or Boehringer Complete protease inhibitor tablets (Roche Applied Science)
  • Beckman ultracentrifuge with TLA 120.2 rotor (or equivalent), and corresponding ultracentrifuge tubes
NOTE: Carry out all procedures at 4°C using solutions prechilled to that temperature.

Support Protocol 2: Immunopurification of SV Subpopulations

  Materials
  • Phosphate‐buffered saline (PBS; appendix 2A) containing 5% (w/v) BSA (fraction V, Boehringer Mannheim)
  • 1000 × protease inhibitors (see recipe) or Boehringer Complete protease inhibitor tablets (Roche Applied Science)
  • Size‐purified synaptic vesicles ( protocol 3 or protocol 4 or protocol 53)
  • Primary antibody to protein of interest (Table 3.12.1)
  • Magnetic bead slurry (Dyanal): e.g., Dynabeads M450 sheep anti‐mouse IgG–coated or Dynabeads M2800 sheep anti‐rabbit IgG–coated beads, depending on species from which primary antibody was derived
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Laemmli sample buffer (unit 17.3)
  • Rotating platform
  • Dynal MPC‐S magnetic stand
  • Beckman tabletop ultracentrifuge with TLA 120.2 rotor (or equivalent), and corresponding polycarbonate ultracentrifuge tubes

Support Protocol 3: Tube Siliconization

  Materials
  • Sigmacote (Sigma)
  • Glass‐ or plasticware to be siliconized
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Figures

Videos

Literature Cited

Literature Cited
   Bajjalieh, S.M., Frantz, G.D., Weimann, J.M., McConnell, S.K., and Scheller, R.H. 1994. Differential expression of synaptic vesicle protein 2 (SV2) isoforms. J. Neurosci. 14:5223‐5235.
   Baumert, M., Maycox, P.R., Navone, F., De Camilli, P., and Jahn, R. 1989. Synaptobrevin: An integral membrane protein of 18,000 daltons present in small synaptic vesicles of rat brain. EMBO J. 8:379‐384.
   Buckley, K. and Kelly, R.B. 1985. Identification of a transmembrane glycoprotein specific for secretory vesicles of neural and endocrine cells. J. Cell Biol. 100:1284‐1294.
   Buckley, K.M., Schweitzer, E.S., Miljanich, G.P., Clift‐O'Grady, L., Kushner, P.D., Reichardt, L.F., and Kelly, R.B. 1983. A synaptic vesicle antigen is restricted to the junctional region of the presynaptic plasma membrane. Proc. Natl. Acad. Sci. U.S.A. 80:7342‐7346.
   Carlson, S.S., Wagner, J.A., and Kelly, R.B. 1978. Purification of synaptic vesicles from elasmobranch electric organ and the use of biophysical criteria to demonstrate purity. Biochemistry 17:1188‐1199.
   Clift‐O'Grady, L., Linstedt, A.D., Lowe, A.W., Grote, E., and Kelly, R.B. 1990. Biogenesis of synaptic vesicle‐like structures in a pheochromocytoma cell line PC‐12. J. Cell Biol. 110:1693‐1703.
   Donovan, J. and Brown, P. 1995. Euthanasia. In Current Protocols in Immunology (J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, and W. Strober, eds.) pp. 1.8.1‐1.8.4. John Wiley & Sons, Hoboken, N.J.
   Floor, E. and Feist, B.E. 1989. Most synaptic vesicles isolated from rat brain carry three membrane proteins, SV2, synaptophysin, and p65. J. Neurochem. 52:1433‐1437.
   Floor, E., Schaeffer, S.F., Feist, B.E., and Leeman, S.E. 1988. Synaptic vesicles from mammalian brain: Large‐scale purification and physical and immunochemical characterization. J. Neurochem. 50:1588‐1596.
   Huttner, W.B., Schiebler, W., Greengard, P., and 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.
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   Janz, R. and Sudhof, T.C. 1999. SV2C is a synaptic vesicle protein with an unusually restricted localization: Anatomy of a synaptic vesicle protein family. Neuroscience 94:1279‐1290.
   Karunanithi, S., Marin, L., Wong, K., and Atwood, H.L. 2002. Quantal size and variation determined by vesicle size in normal and mutant Drosophila glutamatergic synapses. J. Neurosci. 22:10267‐10276.
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   Murthy, V.N. and De Camilli, P. 2003. Cell biology of the presynaptic terminal. Annu. Rev. Neurosci. 26:701‐28.
   Nagy, A., Baker, R.R., Morris, S.J., and Whittaker, V.P. 1976. The preparation and characterization of synaptic vesicles of high purity. Brain Res. 109:285‐309.
   Salazar, G., Love, R., Werner, E., Doucette, M.M., Cheng, S., Levey, A., and Faundez, V. 2004. The zinc transporter ZnT3 interacts with AP‐3 and it is preferentially targeted to a distinct synaptic vesicle subpopulation. Mol. Biol. Cell. 15:575‐587.
   Takamori, S., Rhee, J.S., Rosenmund, C., and Jahn, R. 2000a. Identification of a vesicular glutamate transporter that defines a glutamatergic phenotype in neurons. Nature 407:189‐194.
   Takamori, S., Riedel, D., and Jahn, R. 2000b. Immunoisolation of GABA‐specific synaptic vesicles defines a functionally distinct subset of synaptic vesicles. J. Neurosci. 20:4904‐4911.
   van de Goor, J., Ramaswami, M., and Kelly, R. 1995. Redistribution of synaptic vesicles and their proteins in temperature‐sensitive shibire(ts1) mutant Drosophila. Proc. Natl. Acad. Sci. U.S.A. 92:5739‐5743.
   Wagner, J.A., Carlson, S.S., and Kelly, R.B. 1978. Chemical and physical characterization of cholinergic synaptic vesicles. Biochemistry 17:1199‐1206.
   Whittaker, V.P., Essman, W.B., and Dowe, G.H. 1972. The isolation of pure cholinergic synaptic vesicles from the electric organs of elasmobranch fish of the family Torpedinidae. Biochem J. 128:833‐45.
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