Preparation of Synaptoneurosomes from Mouse Cortex using a Discontinuous Percoll-Sucrose Density Gradient
Summary
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
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
1. Preparations1-4 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. 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. Prepare 10x Stimulation Buffer by making a solution of 100 mM Tris (…
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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.).
Materials
| Name of the reagent | Company | Catalogue number | Comments (optional) |
|---|---|---|---|
| Micro BCA Protein Assay Kit | Pierce | 23235 | |
| CaCl2 | Fisher | C79-500 | |
| CO2 gas | Airgas (UW-MDS) | CD 50 | |
| EDTA | RPI | E57020 | |
| EtOH | Fisher | A407SK-4 | |
| HCl | Fisher | A142-212 | |
| Percoll | GE Healthcare | 17-0891-01 | |
| KH2PO4 | Fisher | P285-500 | |
| PierceSDS-PAGE Sample Prep Kit | Pierce | 89888 | |
| NaCl | RPI | S23020 | |
| NaHCO3 | Fisher | BP328-500 | |
| Na2PHO4 | Fisher | S381-500 | |
| Sucrose | RPI | S24060 | |
| Tris Base | RPI | T60040 | |
| Tetrodotoxin | Sigma | T5651 | |
| Express Pro Label Mix S35 Easy Tag | Perkin Elmer | NEG772 | |
| Equipment | Company | Catalogue number | Comments (optional) |
| Dissection tools | |||
| Dounce homogenizer, 7 mL (comes with two glass pestles labled ” A” and “B”) | Wheaton | ||
| P1000 Gilson Pipetman | Gilson | F123602 | |
| Allegra 6KR Centrifuge | Beckman Coulter | 366830 | |
| GH 3.8 Rotor, Swinging bucket rotor | Beckman Coulter | 360581 | |
| Beckman J2-21 Centrifuge | Beckman | ||
| Beckman tubes with caps | Beckman | 355672 | |
| White walled adapters | Beckman | 342327 | |
| Blue walled adapters | Beckman | ||
| JA-17 Rotor, Fixed-angle rotor | Beckman | 369691 |
Table of antibodies used for western blots:
| Name of Antibody | Company | Catalogue number | Host Species | Dilution Factor |
|---|---|---|---|---|
| β-Actin | Sigma | A5441 | mouse | 1:2000 |
| GFAP | Santa Cruz | sc-65343 | mouse | 1:200 |
| GP73 | Santa Cruz | Sc-134509 | rabbit | 1:200 |
| HSC70 | Santa Cruz | sc-7298 | mouse | 1:200 |
| Laminβ | Santa Cruz | Sc-6261 | goat | 1:200 |
| Prohibitin | Santa Cruz | sc-28259 | rabbit | 1:200 |
| PSD95 | Millipore | MAB1596 | mouse | 5 μg/μL |
| SNAP25 | AbCam | ab5666-100 | rabbit | 1:2000 |
| Synaptophysin | Millipore | MAB368 | mouse | 1:500 |
| β-3-Tubulin | Santa Cruz | sc-80016 | mouse | 1:200 |
References
- Westmark, C. J., Malter, J. S. FMRP mediates mGluR5-dependent translation of amyloid precursor protein. PLoS Biol. 5, e52-e52 (2007).
- 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 protein synthesis. Sci Signal. 3, ra18-ra18 (2010).
- Dunkley, P. R., Heath, J. W., Harrison, S. M., Jarvie, P. E., Glenfield, P. J., Rostas, J. A. 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 (1988).
- Dunkley, P. R., Jarvie, P. E., Robinson, P. J. A rapid Percoll gradient procedure for preparation of synaptosomes. Nat. Protoc. 3, 1718-1728 (2008).
- Hollingsworth, E. B., McNeal, E. T., Burton, J. L., Williams, R. J., Daly, J. W., Creveling, C. R. Biochemical characterization of a filtered synaptoneurosome preparation from guinea pig cerebral cortex: cyclic adenosine 3′:5′-monophosphate-generating systems, receptors, and enzymes. J. Neurosci. 5, 2240-2253 (1985).
- Yi, H., Leunissen, J., Shi, G., Gutekunst, C., Hersch, S. A novel procedure for pre-embedding double immunogold-silver labeling at the ultrastructural level. J. Histochem. Cytochem. 49, 279-284 (2001).
- Akins, M. R., Berk-Rauch, H. E., Fallon, J. R. Presynaptic translation: stepping out of the postsynaptic shadow. Front. Neural Circuits. 3, (2009).
- Jin, I., Kandel, E. R., Hawkins, R. D. Whereas short-term facilitation is presynaptic, intermediate-term facilitation involves both presynaptic and postsynaptic protein kinases and protein synthesis. Learn. Mem. 18, 96-102 (2011).
- Lyles, V., Zhao, Y., Martin, K. C. Synapse formation and mRNA localization in cultured Aplysia neurons. Neuron. 49, 349-356 (2006).
- Wagatsuma, A., Azami, S., Sakura, M., Hatakeyama, D., Aonuma, H., Ito, E. De Novo synthesis of CREB in a presynaptic neuron is required for synaptic enhancement involved in memory consolidation. J. Neurosci. Res. 84, 954-9560 (2006).
- Sutton, M. A., Schuman, E. M. Local translational control in dendrites and its role in long-term synaptic plasticity. J. Neurobiol. 64, 116-131 (2005).
- Li, K. -. W., Hornshaw, M. P., Van der Schors, R. C., Watson, R., Tate, S., Casetta, B., Jimenez, C. R. Proteomics analysis of rat brain postsynaptic density: implications of the diverse protein functional groups for the integration of synaptic physiology. J. Biol. Chem. 279, 987-1002 (2004).
- Peng, J., Kim, M. J., Cheng, D., Duong, D. M., Gygi, S. P., Sheng, M. Semi-quantitative proteomic analysis of rat forebrain postsynaptic density fractions by mass spectrometry. J. Biol. Chem. 279, 21003-21011 (2004).
- Kim, S. H., Fraser, P. E., Westaway, D., St. George-Hyslop, P. H. Group II metabotropic glutamate receptor stimulation triggers production and release of Alzheimer’s amyloid β42 from isolated intact nerve terminals. J. Neurosci. 30, 3870-3875 (2010).
- Weiler, I. J., Greenough, W. T. Potassium ion stimulation triggers protein translation in synaptoneurosomal poiyribosomes. Mol. Cell. Neurosci. 2, 305-314 (1993).
- Billard, J. M. Ageing, hippocampal synaptic activity and magnesium. Magnes Res. 19, 199-215 (2006).
- Murphy, G. G., Fedorov, N. B., Giese, K. P., Ohno, M., Friedman, E., Chen, R., Silva, A. J. Increased neuronal excitability, synaptic plasticity, and learning in aged Kvbeta1.1 knockout mice. Curr. Biol. 14, 1907-1915 (2004).
- Norris, C. M., Halpain, S., Foster, T. C. Reversal of Age-Related Alterations in Synaptic Plasticity by Blockade of L-Type Ca21 Channels. J. Neurosci. 18, 3171-3179 (1998).
- Sallert, M., Malkki, H., Segersträlea, M., Tairaa, T., Lauri, S. E. Effects of the kainate receptor agonist ATPA on glutamatergic synaptic transmission and plasticity during early postnatal development. Neuropharm. 52, 1354-1365 (2007).
- Quinlan, E. M., Philpot, B. D., Huganir, R. L., Bear, M. F. Rapid, experience-dependent expression of synaptic NMDA receptors in visual cortex in vivo. Nat. Neurosci. 2, 357-35 (1999).
- Scheetz, A. J., Nairn, A. C., Constantine-Patton, M. NMDA receptor-mediated control of proteins synthesis at developing synapses. Nat. Neurosci. 3, 211-216 (2000).
- Villasana, L. E., Klann, E., Tejada-Simon, M. V. Rapid isolation of synaptoneurosomes and postsynaptic densities from adult mouse hippocampus. J. Neurosci. Methods. 158, 30-36 (2006).
- Weiler, I. J., Greenough, W. T. Metabotropic glutamate receptors trigger postsynaptic protein synthesis. Proc. Natl. Acad. Sci. U.S.A. 90, 7168-7171 (1993).
- Gray, E. G., Whittaker, V. P. The isolation of nerve endings from brain: An electron microscopic study of cell fragments derived by homogenization and centrifugation. J. Anat. 96, 79-88 (1962).
- Gylys, K. H., Fein, J. A., Yang, F., Cole, G. M. Enrichment of Presynaptic and Postsynaptic Markers by Size-Based Gating Analysis of Synaptosome Preparations From Rat and Human Cortex. Cytometry A. 60, 90-96 (2004).
- Hajós, F. An improved method for the preparation of synaptosomal fractions in high purity. Brain Res. 93, 485-489 (1975).
- Bai, F., Witzmann, F. A. Synaptosome Proteomics. Subcell. Biochem. 43, 77-98 (2007).
- Rao, A., Steward, O. Evidence that protein constituents of postsynaptic membrane specializations are locally synthesized: analysis of proteins synthesized within synaptosomes. J Neurosci. 11, 2881-2895 (1991).
- Pertoft, H., Laurent, T. C., Catsimpoolas, N. Isopycnic separation of cells and cell organelles by centrifugation and modified colloidal silica gradients. Methods of Cell Separation. , 25-65 (1977).
- Ramarao, C. S., Acharya, S. R., Krishnan, K. S., Kenkare, U. W. High affinity uptake of L-glutamate and γ-aminobutyric acid in Drosophila melanogaster. J. Biosci. 11, 119-135 (1987).
- Munteanu, L. S., Dinu, A. Fractionation of granulocytes from whole human blood by centrifugation. Practical hints. Romanian J. Biophys. 14, 53-58 (2004).
- Vlasselaer, P., Van Palathumpat, V. Cell separation composition, kit and method. US patent. , (1997).
Tags
SynaptoneurosomesMouse CortexDiscontinuous Percoll-Sucrose Density GradientHomogenizationFractionationResealed VesiclesIsolated TerminalsSynaptic TransmissionPre- And Postsynaptic CharacteristicsNeuronal SignalingNeurotransmittersFicollCytotoxicCentrifugationFiltrationMechanical DamagePercoll-sucrose Gradient MethodTranslationally Active SNsIsotonic Conditions
Cite This Article
Westmark, P. R., Westmark, C. J., Jeevananthan, A., Malter, J. S. Preparation of Synaptoneurosomes from Mouse Cortex using a Discontinuous Percoll-Sucrose Density Gradient. J. Vis. Exp. (55), e3196, doi:10.3791/3196 (2011).