Neuroscience > Cellular and Developmental Neuroscience

User Ratings

Your rating: None
Your rating: None
Your rating: None (1 vote)
 Add your comments

Multidisciplinary Approaches for Characterizing Synaptic Vesicle Proteins

Miriam Leenders1,  Claudia Gerwin1,  Zu‐Hang Sheng1

1National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland

Publication Name: 
Current Protocols in Neuroscience
Unit Number: 
Unit 2.7
DOI: 
10.1002/0471142301.ns0207s28
Online Posting Date: 
September, 2004
GO TO THE FULL TEXT:
PDF or HTML at Wiley Online Library
Are you the author of this protocol? Login or register and return to this page.

Abstract

Investigation of synaptic vesicle membrane proteins using multidisciplinary approaches, particularly to characterize synaptic vesicle proteins in synapses, can greatly advance our knowledge of the molecular mechanisms involved in synaptic vesicle exocytosis and neurotransmission. Three approaches are presented in this unit to identify and characterize synaptic vesicle proteins. The first is a subcellular fractionation method used to isolate synaptic vesicles from rat brain synaptosomal preparations, which can then be used in a variety of biochemical studies on synaptic vesicle proteins. The second is a detailed procedure for pre-embedding immunogold staining and electron microscopic observation, techniques that permit the morphological identification of synaptic vesicle proteins in individual vesicles at the intact synapse. Finally, a protocol for immunocytochemical staining of cultured hippocampal neurons for light microscopic examination is provided, which allows one to stain multisynaptic vesicle proteins and determine their localization in relation to other proteins or subcellular structures in synapses.

Keywords: Synaptosomes; hippocampal neurons; synapses; light-microscopy; electron-microscopy; immunocytochemistry; immunoblotting

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Unit Introduction
  • Basic Protocol 1: Subcellular Fractionation of Rat Brain Synaptosomes
  • Basic Protocol 2: Visualization of Synaptic Vesicle Proteins by Pre-Embedding Immunogold Electron Microscopy
  • Support Protocol 1: Isolation of Synaptosomes from Rat Brain Cortex
  • Basic Protocol 3: Immunofluorescence Staining of Synaptic Vesicle Proteins on Cultured Hippocampal Neurons
  • Support Protocol 2: Culture of Hippocampal Neurons from Embryonic Rat
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Subcellular Fractionation of Rat Brain Synaptosomes

 Materials
  • Rat brain synaptosomes (see Support Protocol 1)
  • Medium L (see recipe)
  • 1 M KOH
  • 1.0 M sucrose in Medium L (store up to 5 days at 4°C)
  • 20 mM Tris·Cl, pH 7.4 (appendix 2A) containing protease inhibitors (see recipe)
  • Protease inhibitors (see recipe)
  • Sucrose gradients (see recipe)
  • Synaptic vesicle protein–specific antibodies for immunoblotting
  • 55-ml Potter-Elvehjem homogenizer (Teflon-glass)
  • Ultracentrifuge
  • Ultracentrifuge rotors: swinging bucket rotor (e.g., Beckman SW 28) and fixed-angle rotor (e.g., Beckman 70.1 Ti or 50 Ti) capable of 100,000 × g
  • Ultracentrifuge tubes for SW 28 rotor: thick-wall polycarbonate tubes, 31-ml capacity, 25 × 89–mm; and ultraclear (thin-walled) tubes, 38.5-ml capacity, 25 × 89–mm
  • Ultracentrifuge tubes for 70.1 Ti rotor: thick-wall polycarbonate tubes, 10-ml capacity, 16 × 76–mm
  • 10,000 MWCO dialysis tubing
  • Centriprep 10 centrifugal concentrators (Amicon)
  • Additional reagents and equipment for determining protein concentration (cpmb unit 10.1A), SDS-PAGE (cpmb unit 10.2), and immunoblotting (cpmb unit 10.8)

NOTE: All fractionation steps must be performed between 0° to 4°C, and all solutions, centrifuge tubes, and centrifuge rotors should be precooled below 4°C and kept on ice.

Basic Protocol 2: Visualization of Synaptic Vesicle Proteins by Pre-Embedding Immunogold Electron Microscopy

 Materials
  • Rat brain synaptosomes (see Support Protocol 1)
  • Wash buffer (see recipe)
  • 0.1 M sodium phosphate buffer (see recipe)
  • 4% (w/v) paraformaldehyde in 0.1 M sodium phosphate buffer
  • 5% (v/v) normal goat serum/0.1% (w/v) saponin in PBS (see appendix 2A for PBS)
  • Primary antibody against synaptic vesicle protein of interest
  • PBS (appendix 2A) with and without 5% (w/v) nonfat dry milk
  • Nanogold-conjugated secondary antibody (Nanoprobes)
  • 2% (w/v) glutaraldehyde in PBS (see appendix 2A for PBS)
  • HQ silver enhancement kit (Nanoprobes)
  • 12-well tissue culture plates
  • Electron microscope
  • Additional reagents and equipment for preparing samples for electron microscopy (see unit 1.2)

NOTE: All steps are performed at room temperature.

Support Protocol 1: Isolation of Synaptosomes from Rat Brain Cortex

 Materials
  • 3- to 4-week old male rat (Wistar or Sprague-Dawley)
  • 1× sucrose buffer (see recipe), ice-cold
  • Percoll gradients (see recipe), ice-cold
  • Wash buffer (see recipe), ice-cold
  • Rat guillotine
  • 25-ml Potter-Elvehjem homogenizer (Teflon-glass)
  • High-speed centrifuge (e.g., Sorvall RC-5C with SS-34 rotor and adaptors for eight 16 × 100–mm tubes)
  • Polycarbonate high-speed centrifuge tubes: 11-ml capacity, 16 × 100–mm and 50-ml capacity, 29 × 102–mm
  • Additional reagents and equipment for anesthesia of the rat (appendix 4B)

NOTE: All isolation steps must be performed between 0° to 4°C, and all solutions, centrifuge tubes, and centrifuge rotors should be precooled below 4°C and kept on ice.

Basic Protocol 3: Immunofluorescence Staining of Synaptic Vesicle Proteins on Cultured Hippocampal Neurons

 Materials
  • Hippocampal neurons cultured on coverslips (see Support Protocol 2)
  • Phosphate-buffered saline (PBS; appendix 2A), pH 7.4
  • Fixative solution: 4% (w/v) paraformaldehyde/4% (w/v) sucrose in PBS
  • Blocking/permeabilization buffer (see recipe)
  • Primary antibody against protein interest
  • Antibody dilution buffer (see recipe)
  • Secondary antibody labeled with fluorophore (e.g., TRITC, FITC, Cy2, Cy3, or Cy5; store protected from light)
  • Mounting medium
  • 12-well tissue culture plates
  • Microscope slides
  • Forceps
  • Fluorescence microscope with 40× and/or 63× oil-immersion lenses

NOTE: All steps are performed at room temperature.

Support Protocol 2: Culture of Hippocampal Neurons from Embryonic Rat

 Materials
  • 19-day pregnant rat
  • 70% (v/v) ethanol
  • Dissection buffer (see recipe), ice-cold
  • Chopping buffer (see recipe), ice-cold
  • Trypsin (Invitrogen)
  • Glial feed (see recipe), with and without GlutaMAX supplement
  • Neuronal feed (see recipe)
  • Surgical instruments:
    • Straight surgical scissors
    • Straight fine scissors
    • Straight spring scissors
    • Curved forceps
    • Straight forceps
    • Dumont no. 5-45 forceps (45° tip)
  • 35-mm tissue culture dishes
  • Dissecting microscope and fiber-optic lights
  • 15-ml tubes
  • 9-in. cotton-plugged borosilicate glass pipets with flame-polished tips
  • Polyornithine/fibronectin-coated (unit 3.2) 12- to 15-mm-diameter circular coverslips and/or two-chamber Lab-Tek slides (Nalge Nunc International)
  • Additional reagents and equipment for anesthesia of the rat (appendix 4B) and tissue culture (appendix 3B)

NOTE: All solutions and equipment coming into contact with living cells must be sterile, and aseptic technique should be used accordingly.

NOTE: All culture incubations should be performed in a humidified, 37°C, 5% CO2 incubator unless otherwise specified.


Looking for materials?  Suppliers Guide
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

  • Figure 2.7.1
    Flow chart for fractionation of synaptosomes.

    View Image
  • Figure 2.7.2
    SDS-PAGE of subcellular fractions from rat brain synaptosomes. Crude synaptosomes were sedimented from rat brain homogenate by differential centrifugation, and separated into fractions enriched in presynaptic cytosol (PC), synaptic vesicles (SV), and synaptosome plasma membrane (PM). Equal amounts (8 µg) of synaptosome fractions and 50 µg of crude synaptosomes were analyzed by SDS-PAGE and sequentially immunoblotted with antibodies as indicated on the same blot membrane. The relative purity of subcellular fractions was determined by the markers for synaptic vesicle (VAMP2 and synaptophysin), plasma membrane (Na/K-ATPase), and cytosol (lactate dehydrogenase LDH).

    View Image
  • Figure 2.7.3
    Immunogold electron micrographs of synaptosomes isolated from adult rat brain. Synaptophysin staining was visualized by immunogold labeling followed by silver enhancement (see Basic Protocol 2). Gold particles (dark) label synaptophysin on synaptic vesicles. Scale bars: 100 nm.

    View Image
  • Figure 2.7.4
    Immunofluorescent staining of cultured hippocampal neurons. Cultured hippocampal neurons from E18 rat embryos were fixed at day in vitro (DIV) 35, stained with anti-synaptophysin antibody, and visualized by rhodamine (TRITC)-coupled secondary antibody (see Basic Protocol 3). Scale bar: 10 µm.

    View Image
  • Figure 2.7.5
    Illustration of rat brain anatomical features mentioned in Support Protocols 1 and 2.

    View Image

Literature Cited

Literature Cited
    Betz, W.J., Mao, F., and Bewick, G.S. 1992. Activity-dependent fluorescent staining and destaining of living vertebrate motor nerve terminals. J. Neurosci. 12:363-375.
    Betz, W.J., Mao, F., and Smith, C.B. 1996. Imaging exocytosis and endocytosis. Curr. Opin. Neurobiol. 6:365-371.
    Booth, R.F.G. and Clark, J.B. 1978. A rapid method for the preparation of relatively pure metabolically competent synaptosomes from rat brain. Biochem. J. 176:365-370.
    Breukel, A.I.M., Besselsen, E., and Ghijsen, W.E.J.M. 1997. Synaptosomes: A model system to study release of multiple classes of neurotransmitters. In Methods in Molecular Biology, vol. 72, Neurotransmitter Methods (R.C. Rayne, ed.) pp. 33-47. Humana Press, Totowa, N.J.
    Cousin, M.A. and Robinson, P.J. 1999. Mechanisms of synaptic vesicle recycling illuminated by fluorescent dyes. J. Neurochem. 73:2227- 2239.
    Cuello, A.C. 1993. Immunohistochemistry II. IBRO Handbook Series, Methods in the Neurosciences, John Wiley & Sons, Chichester, U.K.
    Dagani, F., Zanada, F., Marzatico, F., and Benzi, G. 1985. Free mitochondria and synaptosomes from single rat forebrain: A comparison between two known subfractionating techniques. J. Neurochem. 45:653-656.
    Das, S., Boczan, J., Gerwin, C.M., Zald, P., and Sheng, Z.-H. 2003. Regional and developmental regulation of syntaphilin expression in the brain: A candidate molecular element of synaptic functional differentiation. Mol. Brain Res. 116:38-49.
    De Camilli, P., Haucke, V., Takei, K., and Mugnaini, E. 2000. The structure of synapses. In Synapse (W.M. Cowan, T.C. Sudhof, and C.F. Stevens, eds.) pp. 89-134. Johns Hopkins University Press, Baltimore, Md.
    Dunkley, P.R., Heath, J.W., Harrison, S.M., Jarvie, P.E., Glenfield, P.J., and Rostas, J.A.P. 1988. 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.
    Fernandez-Chacon, R. and Südhof, T.C. 1999. Genetics of synaptic vesicle function: Toward the complete functional anatomy of an organelle. Annu. Rev. Physiol. 61:753-776.
    Gray, E.G. and Whittaker, V.P. 1962. The isolation of nerve endings from brain: An electron microscopic study of cell fragments derived by homogenization and centrifugation. J. Anat. 96:82-83.
    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.
    Ilardi, J.M., Mochida, S., and Sheng, Z.-H. 1999. Snapin: A SNARE-associated protein implicated in synaptic transmission. Nat. Neurosci. 2:119-124.
    Lao, G., Scheuss, V., Gerwin, C.M., Su, Q., Mochida, S., Rettig, J., and Sheng, Z.-H. 2000. Syntaphilin: A syntaxin-1 clamp that controls SNARE assembly. Neuron 25:191-201.
    Nagy, A. and Delgado-Escueta, A.V. 1984. Rapid preparation of synaptosomes from mammalian brain using nontoxic isoosmotic gradient material (Percoll). J. Neurochem. 43:1114-1123.
    Su, Q., Mochida, S., Tian, J.-H., Mehta, R., and Sheng, Z.-H. 2001. SNAP-29: A general SNARE protein that inhibits SNARE disassembly and is implicated in synaptic transmission. Proc. Natl. Acad. Sci. U.S.A. 98:1438-1443.
    Tanner, V.A., Ploug, T., and Tao-Cheng, J.-H. 1996. Subcellular localization of SV2 and other secretory vesicle components in PC12 cells by an efficient method of pre-embedding EM immunocytochemistry for cell cultures. J. Histochem. Cytochem. 44:1481-1488.
    Tian, J.-H., Das, S., and Sheng, Z.-H. 2003. Ca+-dependent phosphorylation of syntaxin-1A by DAP-kinase regulates its interaction with Munc-18. J. Biol. Chem. 278:26265-26274.
    Whittaker, V.P. 1993. Thirty years of synaptosome research. J. Neurocytol. 22:735-742.
    Zhai, R.G., Vardinon-Friedman, H., Cases-Langhoff, C., Becker, B., Gundelfinger, E.D., Ziv, N.E., and Garner, C.C. 2001. Assembling the presynaptic active zone: A characterization of an active zone precursor vesicle. Neuron 29:131-143.
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library
Looking for Answers?
Do you have tips, tricks, or improvements to share?

Join the Conversation

Post new comment

The content of this field is kept private and will not be shown publicly.
CAPTCHA
This question is for testing whether you are a human visitor and to prevent automated spam submissions.