Method Article

Preparation of Synaptoneurosomes from Mouse Cortex using a Discontinuous Percoll-Sucrose Density Gradient

DOI:

10.3791/3196

⸱

September 17th, 2011

 ,  ,  , 
1Department of Pathology and Laboratory Medicine, Waisman Center for Developmental Disabilities, University of Wisconsin, 2Department of Biochemistry, Waisman Center for Developmental Disabilities, University of Wisconsin

In This Article

Summary

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A method to prepare translationally active, intact synaptoneurosomes (SNs) from mouse brain cortex is described. The method uses a discontinuous Percoll-sucrose density gradient allowing for the quick preparation of active SNs.

Abstract

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Synaptoneurosomes (SNs) are obtained after homogenization and fractionation of mouse brain cortex. They are resealed vesicles or isolated terminals that break away from axon terminals when the cortical tissue is homogenized. The SNs retain pre- and postsynaptic characteristics, which makes them useful in the study of synaptic transmission. They retain the molecular machinery used in neuronal signaling and are capable of uptake, storage, and release of neurotransmitters.

The production and isolation of active SNs can be problematic using medias like Ficoll, which can be cytotoxic and require extended centrifugation due to high density, and filtration and centrifugation methods, which can result in low activity due to mechanical damage of the SNs. However, the use of discontinuous Percoll-sucrose density gradients to isolate SNs provides a rapid method to produce good yields of translationally active SNs. The Percoll-sucrose gradient method is quick and gentle as it employs isotonic conditions, has fewer and shorter centrifugation spins and avoids centrifugation steps that pellet SNs and cause mechanical damage.

Protocol

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1. Preparations1-4

  1. Prepare 500 mL of gradient medium (GM) buffer by mixing 50 mL of a 2.5 M sucrose slurry, 2.5 mL of a 1M Tris-HCl, pH7.5 stock, and 0.1 mL of a 0.5 m EDTA, pH 8.0 stock with milli-Q water to volume. Filter sterilize the solution, aliquot and store frozen.
  2. Prepare a 1000x stock of tetrodotoxin (TTX) by making a 1 mM solution in milli-Q H2O. Aliquots of TTX can be stored at -20°C.
  3. Prepare 10x Stimulation Buffer by making a solution of 100 mM Tris (pH 7.5), 5 mM Na2HPO4, 4 mM KH2PO4, 40 mM NaHCO3, and 800 mM NaCl in milli-Q water. Stock can be st....

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Discussion

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The discontinuous Percoll-sucrose gradient preparation described herein is a quick, reliable method to isolate active SNs, which can be used in a variety of synaptic transmission experiments. This gradient method, which is based on the method developed by Dunkley et al.,3,4 is a subcellular brain fractionation procedure that isolates both pre- and postsynaptic membrane-derived vesicles that are associated with one another. Without further purification these SNs are compatible with a variety of molecular biolog.......

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Disclosures

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No conflicts of interest declared.

Acknowledgements

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We would like to thank B. K. August from the University of Wisconsin-Madison Electron Microscope Facility for the electron microscopy. This work was supported by NIH grants R01-DA026067 and P30-HD03352 (to J.S.M.).

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Name of the reagent Company Catalogue number Comments (optional)
Micro BCA Protein Assay KitPierce23235 
CaCl2FisherC79-500 
CO2 gasAirgas (UW-MDS)CD 50 
EDTARPIE57020 
EtOHFisherA407SK-4 
HClFisherA142-212 
PercollGE Healthcare17-0891-01 
KH2PO4FisherP285-500 
PierceSDS-PAGE Sample Prep KitPierce89888 
NaClRPIS23020 
NaHCO3FisherBP328-500 
Na2PHO4FisherS381-500 
SucroseRPIS24060 
Tris BaseRPIT60040 
TetrodotoxinSigmaT5651 
Express Pro Label Mix S35 Easy TagPerkin ElmerNEG772 
    
Equipment Company Catalogue number Comments (optional)
Dissection tools   
Dounce homogenizer, 7 mL (comes with two glass pestles labled " A" and "B")Wheaton  
P1000 Gilson PipetmanGilsonF123602 
Allegra 6KR CentrifugeBeckman Coulter366830 
GH 3.8 Rotor, Swinging bucket rotorBeckman Coulter360581 
Beckman J2-21 CentrifugeBeckman  
Beckman tubes with capsBeckman355672 
White walled adaptersBeckman342327 
Blue walled adaptersBeckman  
JA-17 Rotor, Fixed-angle rotorBeckman369691 

Table of antibodies used for western blots:

Name of Antibody Company Catalogue number Host Species
β-ActinSigmaA5441mouse
GFAPSanta Cruzsc-65343mouse
GP73Santa CruzSc-134509rabbit
HSC70Santa Cruzsc-7298mouse
LaminβSanta CruzSc-6261goat
ProhibitinSanta Cruzsc-28259rabbit
PSD95MilliporeMAB1596mouse
SNAP25AbCamab5666-100rabbit
SynaptophysinMilliporeMAB368mouse
β-3-TubulinSanta Cruzsc-80016mouse

References

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  1. Westmark, C. J., Malter, J. S. FMRP mediates mGluR5-dependent translation of amyloid precursor protein. PLoS Biol. 5, e52-e52 (2007).
  2. Westmark, P. R., Westmark, C. J., Wang, S., Levenson, J., O'Riordan, K. J., Burger, C., Malter, J. S. Pin1 and PKMzeta sequentially control dendritic pr....

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Tags

Percoll Sucrose GradientDensity Gradient CentrifugationSynaptosome IsolationWestern Blot AnalysisS35 Methionine IncorporationProtein Translation StudiesSynaptic Vesicle UptakeNeurotransmitter Release

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