Characterizing Synaptic Vesicle Proteins Using Synaptosomal Fractions and Cultured Hippocampal Neurons

Jerome DiGiovanni1, Tao Sun1, Zu‐Hang Sheng2

1 These authors contributed equally to this work, null, null, 2 National 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.ns0207s59
Online Posting Date:  April, 2012
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Abstract

Cloning and characterization of synaptic vesicle proteins and their binding counterparts on the presynaptic plasma membrane have greatly advanced our understanding of the molecular mechanisms involved in the synaptic vesicle cycle and neurotransmitter release. This unit discusses multidisciplinary approaches to characterize proteins from synaptosome‐enriched subcellular fractions and localize them within cultured neurons. The first approach regroups methods used to isolate synaptic vesicles from rat brain synaptosomal preparations, allowing for specific biochemical investigation of synaptic vesicle proteins. The second is a detailed procedure for pre‐embedding immunogold staining and electron microscopic observation, which permits the morphological identification of proteins in individual vesicles at intact synapses. Additionally, this chapter proposes methods for light microscopic examination of hippocampal neurons. It includes procedures for embryonic and postnatal hippocampal neuron culture and describes an immunocytochemical staining protocol used to investigate synaptic vesicle protein localization with respect to other proteins or subcellular structures. Curr. Protoc. Neurosci. 59:2.7.1‐2.7.22. © 2012 by John Wiley & Sons, Inc.

Keywords: synaptosomes; hippocampal neurons; synapses; light microscopy; electron microscopy; immunocytochemistry; immunoblotting

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation and Subcellular Fractionation of Rat Brain Synaptosomes
  • Basic Protocol 2: Visualization of Synaptic Vesicle Proteins by Pre‐Embedding Immunogold Electron Microscopy
  • Basic Protocol 3: Immunofluorescence Staining of Synaptic Vesicle Proteins in Cultured Hippocampal Neurons
  • Support Protocol 1: Culture of Hippocampal Neurons from Postnatal Rats or Mice
  • Support Protocol 2: Culture of Hippocampal Neurons from Embryonic Rats or Mice
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Isolation and Subcellular Fractionation of Rat Brain Synaptosomes

  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
  • Medium L (see recipe)
  • 1 M KOH
  • 1.0 M sucrose in Medium L (store up to 5 days at 4°C)
  • Sucrose gradients (see recipe) in thin‐walled ultracentrifuge tubes (ultraclear tubes, 38.5‐ml capacity, 25 × 89 mm)
  • Protease inhibitors (see recipe)
  • 20 mM Tris⋅Cl, pH 7.4 ( appendix 2A)
  • Antibodies specific for subcellular fractions: e.g., anti‐synaptophysin (Chemicon) or anti‐VAMP (StressGen) for synaptic vesicles, anti‐Na+/K+‐ATPase (Transduction Laboratories) for plasma membranes, and anti−lactate dehydrogenase (anti‐LDH, Abcam) for cytosol
  • Rat guillotine
  • Surgical tools for isolation of cortex
  • 25‐ and 55‐ml Potter‐Elvehjem homogenizers (Teflon‐glass)
  • High‐speed centrifuge (e.g., Sorvall RC‐5C with SS‐34 rotor and adaptors for eight 16 × 100–mm tubes)
  • 50‐ml (29 × 102−mm) polycarbonate high‐speed centrifuge tubes
  • Ultracentrifuge capable of 100,000 × g, with:
    • Swinging bucket rotor (e.g., Beckman SW28) and 31‐ml, 25 × 89−mm, thick‐walled polycarbonate ultracentrifuge tubes
    • Fixed‐angle rotor (e.g., Beckman 70.1 Ti or 50 Ti) with 10‐ml, 16 × 76−mm thick‐walled polycarbonate ultracentrifuge tubes
  • 10,000 MWCO dialysis membranes
  • Centriprep‐10 centrifugal concentrators (Millipore)
  • Additional reagents and equipment for rat anesthesia ( appendix 4B), dialysis, determining protein concentration (e.g., Simonian and Smith, ), SDS‐PAGE (e.g., Gallagher, ), and immunoblotting (e.g., Gallagher et al., )
NOTE: All isolation steps must be performed at 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
  • Unfractionated rat brain synaptosomes (see protocol 1, step 7)
  • Wash buffer (see recipe)
  • 0.1 M sodium phosphate buffer, pH 7.4 (see recipe)
  • 4% (w/v) paraformaldehyde in 0.1 M sodium phosphate buffer
  • PBS ( appendix 2A)
  • 5% (v/v) normal goat serum/0.1% (w/v) saponin in PBS
  • 5% (w/v) nonfat dry milk in PBS
  • Primary antibody against synaptic vesicle protein of interest
  • Nanogold‐conjugated secondary antibody (Nanoprobes)
  • 2% (w/v) glutaraldehyde in PBS
  • HQ silver enhancement kit (Nanoprobes)
  • Filter paper
  • Razor blade
  • 12‐well tissue culture plates
  • Additional reagents and equipment for preparing samples for electron microscopy (unit 1.2)
NOTE: All steps are performed at room temperature.

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

  Materials
  • Hippocampal neurons cultured on coverslips (see Support Protocols protocol 41 and protocol 52)
  • Phosphate‐buffered saline (PBS; appendix 2A), pH 7.4
  • Fixative solution: 4% (w/v) paraformaldehyde/4% (w/v) sucrose in PBS
  • Blocking/permeabilization buffer: 10% (v/v) normal goat serum/0.2% (v/v) Triton X‐100 or saponin in PBS (prepare fresh)
  • Primary antibody against protein of interest
  • Antibody dilution buffer: 10% (v/v) normal goat serum/0.05% (v/v) Triton X‐100 or saponin in PBS (prepare fresh)
  • Fluorophore‐conjugated secondary antibody (e.g., TRITC, FITC, Cy2, Cy3, or Cy5; store protected from light)
  • Anti‐fading mounting medium (e.g., Fluoroshield, Accurate Chemical & Scientific Corp.)
  • 12‐well tissue culture plates
  • Microscope slides
  • Forceps
  • Fluorescence microscope with 40× and/or 63× oil‐immersion objectives
NOTE: Fluorophores are light‐sensitive reagents. All solutions and stained samples should be protected from light, e.g., using aluminum foil.NOTE: All steps are performed at room temperature.

Support Protocol 1: Culture of Hippocampal Neurons from Postnatal Rats or Mice

  Materials
  • Xylenes
  • 100% (v/v) ethanol
  • Polyornithine (Sigma P4957)
  • Ultrapure water
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Fibronectin (Sigma F1141‐5MG)
  • P0 mouse pups or P2‐3 rat pups
  • Hank's balanced salt solution (HBSS; see recipe)
  • HBSS containing 20% fetal bovine serum (FBS), ice cold, prepare fresh
  • 50 mg/ml trypsin (type XI, Sigma, T1005) in H 2O (store 10‐mg aliquots at −20°C)
  • 20 mg/ml DNase (type IV, Sigma, D5025) in H 2O (store 1‐mg aliquots at −20°C)
  • Digestion buffer (see recipe), ice cold
  • Dissociation buffer: 150 mg MgSO 4⋅7H 2O in 50 ml HBSS (final 12 mM), ice cold, prepare fresh
  • Plating medium (see recipe), 37°C
  • Feeding medium 1 (see recipe) with 3 µM and 6 µM Ara‐C, 37°C
  • 12‐ or 25‐mm‐diameter round coverslips
  • Sonicator
  • Gauze pads
  • Glass Petri dishes
  • 250°F oven
  • Dissecting tools:
    • Spring scissors, straight and angled
    • Forceps, straight and curved
    • Angled dissecting bone chisel
    • Scalpel
    • Straight surgical scissors
  • 35‐ and 60‐mm tissue culture dishes
  • Dissecting microscope with fiber‐optic lights
  • 9‐in. borosilicate glass pipets (Pasteur pipets)
  • Plastic transfer pipets
  • 15‐ml polypropylene (not polycarbonate) conical centrifuge tubes
  • 0.22‐µm filter
  • Fire‐polished, sterile, siliconized/plugged Pasteur pipets, 0.5‐ and 0.75‐mm bore size

Support Protocol 2: Culture of Hippocampal Neurons from Embryonic Rats or Mice

  Materials
  • 18‐ or 19‐day pregnant rat or mouse
  • 70% (v/v) ethanol
  • Dissection buffer (see recipe), ice‐cold
  • Papain solution (see recipe)
  • 1/1 solution (see recipe)
  • 10/10 solution (see recipe)
  • Plating medium 2 (see recipe), 37°C
  • Feeding medium 2 (see recipe), 37°C
  • Dissecting tools:
    • Straight wide‐tipped forceps
    • Straight surgical scissors
    • Straight fine scissors
    • Forceps, curved and straight
    • Spring scissors, straight and angled
    • Dumont forceps (#5/45, 45° angled tip)
  • Dissecting microscope and fiber‐optic lights
  • 35‐ and 100‐mm tissue culture dishes
  • 15‐ml polypropylene (not polycarbonate) conical centrifuge tubes
  • 5‐ml pipet
  • Polyornithine/fibronectin‐coated 12‐ or 25‐mm‐diameter round coverslips (see protocol 4, steps 1‐10)
  • Additional reagents and equipment for rodent anesthesia and euthanasia **( appendix 4B & appendix 4H) and counting cells with a hemacytometer ( appendix 3B)
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Figures

  •   FigureFigure 2.7.1 Illustration of rat brain anatomical features.
  •   FigureFigure 2.7.2 Flow chart for fractionation of synaptosomes.
  •   FigureFigure 2.7.3 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) (see ). Equal amounts (8 µg) of synaptosome fractions and 50 µg of crude synaptosomes were analyzed by SDS‐PAGE and then sequentially immunoblotted with the indicated antibodies. The relative purity of subcellular fractions was determined by staining for markers of synaptic vesicles (VAMP2 and synaptophysin), plasma membrane (Na/K‐ATPase), and cytosol (lactate dehydrogenase, LDH).
  •   FigureFigure 2.7.4 Immunogold electron micrographs of synaptosomes isolated from adult rat brain. Synaptophysin staining was visualized by immunogold labeling followed by silver enhancement (see ). Gold particles (dark) label synaptophysin on synaptic vesicles. Scale bars: 100 nm.
  •   FigureFigure 2.7.5 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‐conjugated secondary antibody (see ). Scale bar: 10 µm.

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