The protocol by Smalla KH et al. (as described in: Neuromethods. The Cytoskeleton. Chapter 11: Isolation of the Postsynaptic Density: A Specialization of the Subsynaptic Cytoskeleton. (2013) 79: pp. 265-280) has been successfully used by myself. Of course, you don't have to go all the way to PSD, but may stop with the synaptosomes. It's sucrose gradient based, but maybe it is different from the protocol you tried at some other point.

Brain Synaptosomal Preparation protocol (work very good)

By Dr. Taylor Kimberly *

1. Homogenize brain tissue. Add 5 ml of 0.32 M sucrose. Homogenize with 12 strokes of a 19x84mm tissue grinder (Potter Elvehjem, plastic coated) at 800 rpm.

2. Centrifuge to remove large debris. Centrifuge for 10 min at 1000 g at RT. Remove 100 ul for synaptophysin analysis.

3. Layer supernatant onto sucrose and centrifuge again.

4. Carefully layer the supernatant onto 4 ml of 1.2 M sucrose in a SW41 centrifuge tube (Beckman).

5. Spin at 160,000 g for 15 min (= 33,000 rpm with SW41 rotor).

6. Carefully remove synaptosome layer. The synaptosomes are at the interface between the 1.2 M and 0.32 M sucrose layers. It is a slightly cloudy thin layer. Mitochondria and lysosomes pellet to the bottom.

7. Remove 100 ul for synaptophysin analysis.

8. Dilute synaptosomes with 0.32 M sucrose. Add 4 ml of 0.32 M sucrose to synaptosomes, mix, and then carefully layer onto 4 ml of 0.8 M sucrose in a fresh centrifuge tube.

9. Centrifuge to pellet synaptosomes.

Spin at 160,000 g for 15 min. The pellet is enriched in synaptosomes.

10. Discard the supernatant and resuspend in 1 ml of 1X STE.

11. Remove 50 ul for electron microscopy analysis.

12. Lyse synaptosomes for immunoprecipitation analysis. Add 110 ul of 10% NP-40 STEN lysis (- BSA) and nutate at 4°C for 20 min. Protein normalize samples, remove sample for synaptophysin analysis then continue IP according to standard protocols.

Buffers:

2.55 M sucrose (unbuffered). Heat on a stirring block to dissolve, then bring up to 1 L. Determine concentration using a refractometer and adjust to 2.55 M sucrose (refractive index is 1.4558 for 87.28% sucrose (w/v) at 2.5498 M)

10% NP-40 STEN lysis buffer (-BSA): 5 ml 2x STEN; 1 ml NP-40; 0.1 ml 100X PI; 0.1 ml 100x Pefabloc; 3.8 ml ddH2O; Store at -20°C.

PLoS One. 2009 Jun 23;4(6):e6021. doi: 10.1371/journal.pone.0006021.

Long-term potentiation in isolated dendritic spines.

Corera AT, Doucet G, Fon EA.

Source

Centre for Neuronal Survival and Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada.

Fallon L, Moreau F, Croft BG, Labib N, Gu WJ, et al. Parkin and CASK/LIN-2 associate via a PDZ-mediated interaction and are co-localized in lipid rafts and postsynaptic densities in brain. J Biol Chem. 2002;277:486–491. [PubMed]

Fallon L, Belanger CM, Corera AT, Kontogiannea M, Regan-Klapisz E, et al. A regulated interaction with the UIM protein Eps15 implicates parkin in EGF receptor trafficking and PI(3)K-Akt signalling. Nat Cell Biol. 2006;8:834–842. [PubMed]

THis is what you're looking for. Good luck!

Curr Protoc Neurosci. 2008 Jan;Chapter 1:Unit 1.16. doi: 10.1002/0471142301.ns0116s42.

Biochemical fractionation of brain tissue for studies of receptor distribution and trafficking.

Hallett PJ, Collins TL, Standaert DG, Dunah AW.

Source

MassGeneral Institute for Neurodegenerative Disease, Massachusetts General Hospital and Harvard Medical School, Charlestown, Massachusetts, USA.

Abstract

An important tool for studying the regulation of synapses is a rapid and reliable means of separating synaptic and intracellular proteins. This unit presents a technique for analysis of brain tissue which relies on differential centrifugation to separate proteins present at synaptic sites from those found in intracellular cytoplasmic and vesicular pools. The method is efficient in that only small amounts of tissue, such as might be obtained from a small region of a rodent brain, are required. It is reproducible and, in conjunction with immunoblot or immunoprecipitation techniques, can produce reliable quantitative data. The protocol will be of interest to those conducting a variety of different studies related to the localization and trafficking of brain receptors and signaling molecules.

Copyright 2008 by John Wiley & Sons, Inc.

PMID: 18428670 [PubMed - indexed for MEDLINE]

Hi Pascal,

are you even sure it's the synaptosomal isolation process that is deficient?

Can you rule out any flaws in your uptake protocoll and your supplies?

It occurs, that the serotonin is to old and therefore degraded or maybe your uptake protocol is not adapted to a putative change of specific activity between two 3H - 5-HT lots of the same provider.

As to the synaptosomes, you can prepare a 15 ml douncer tube with 5 ml of a homogenzation buffer that contains 0.32 mol/l sucrose, 4 mmol/l Hepes (pH 7.4), Protease- Inhibitor Mix and 1mmol/l PMSF,

add your mouses brain (1 brain/5 ml),

potter 9x @ 900 rpm,

spin 10' @ 4°C, 5000 rpm (TLA 110 rotor, Beckman&Coulter),

take off supernatant and spin it 15' @ 16000 rpm (same rotor),

discard this supernatant, This second pellet comes in layers of differentially shaded layers, as you might have already observed. Resuspend the outer bright sediment only, using 500 µl of your uptake buffer. A sodium buffer, I suppose.

This protocol is similar to the Hallett PJ et al. protocol already posted by Stéphane and to Dr. Taylor Kimberly's as posted by Mohammad above.

Gutes Gelingen!

Thanks a lot for these infos ! I'll have a closer look at these protocols.

To Sascha: I'm not sure at all that the problem doesn't come from the uptake protocol, but my 3H-5HT is not so old and works fine for uptake in cell lines or primary neurons. And when I try for synaptosomes I get the same values with various doses of 5HT (no dose-response curve), and with non-specific uptake (with fluoxetine). I must do something wrong...

I myself purified brain synaptosome from both of mouse and sheep whole brain to investigate the synaptosome morphology and AchE activity. I offer you to use Dodd protocol because you can highly purify synaptosomes with a simple surose gradient and it will last less than 2-3 hours. It`s a rapid method. I sent you the corresponding paper and I hope it would be useful for you.

Article A rapid method for preparing synaptosomes: Comparison, with ...

If you have any problem with surose gradient protocol, I introduce a percoll gradient instead. It is a rapid protocol too and my colleague used this protocol and obtained acceptable results.

Article A rapid Percoll gradient procedure for preparation of synaptosomes

You find enclosed some references. We currently prepare mouse synaptosomes to study neurotransmitter release, from selective brain areas, evaluating both the release of prelabelled compounds or of endogenous neurotrasmitters:

Summa M., Di Prisco S., Grilli M., Usai C., Marchi M., Pittaluga A.

Presynaptic mGlu7 receptors controls GABA release in mouse hippocampus.

Neuropharmacology, 66, 215-224, 2013

Grilli M., Zappettini,S., Zanardi A., Lagomarsino F., Pittaluga A., Zoli M., Marchi M.

Exposure to an enriched environment selectively increases the functional response of the pre-synaptic NMDA receptors which modulate noradrenaline release in mouse hippocampus. J. Neurochem., 110, 1598-1606, 2009

Grilli M., Neri E., Zappettini S., Massa F., Bisio A., Romussi G., Marchi M., Pittaluga A., Salvinorin A exerts opposite presynaptic controls on neurotransmitter exocytosis from mouse brain nerve terminals Neuropharmacology, 57, 523–530, 2009

Grilli M., Zappettini S., Zoli M., Marchi M.

Pre-synaptic nicotinic and D2 receptors functionally interact on dopaminergic nerve endings of rat and mouse nucleus accumbens

J. Neurochem. ,108, 1507-1514, 2009

Grilli M., Zappettini S., Raiteri L., Marchi M., Nicotinic and muscarinic cholinergic receptors coexist on GABAergic nerve endings in the mouse striatum and interact in modulating GABA release Neuropharmacology.56, 610 614,2009

The references stated by others may mention this, but you will likely need a functionally "active" synaptosomal preparation, which can be a bit different from traditional synaptic preparations used for biochemical assays. Try looking for "synaptoneurosome" protocols (I know Mike Ehlers has used these). Most of these protocols have an aCSF-like buffer as a basis, but importantly, also have an ATP regenerating system associated, including Mg-ATP, creatine phosphate and creatine phosphokinase. Proper ATP, sodium and potassium levels will probably be important for you given the serotonin transporter ultimately requires Na/K pumps. Good luck.

Dear Pascal,

I usually use the following protocol (sucrose gradient fractionation, similar to that one posted above) for synaptosome preparation, with great results.

Pure synaptosome preparation.

All procedures should be done at 4°C using precooled reagents. For rat and mouse samples, immediately remove brain from the cranium into ice cold HEPES-buffered sucrose (0.32 M sucrose, 4 mM HEPES pH 7.4) containing a freshly added protease inhibitor cocktail and phosphatase inhibitor cocktail (optional).

1. Add 10 volumes of HEPES-buffered sucrose (0.32 M sucrose, 4 mM HEPES pH7.4) to the tissue and homogenize in a motor driven glass-teflon homogenizer at ~900 rpm (10-15 strokes). Never use polytron.

2. Centrifuge the homogenate (Hom.) at 800-1000 x g at 4°C to remove the pelleted nuclear fraction (P1). Note: Save 150 ul aliquot.

3. Spin resulting supernatant (S1) at 10,000 x g for 15 min (9200 rpm in SL50T rotor) to yield the crude synaptosomal pellet (P2).

4. Resuspend pellet (P2) in 10 volumes of HEPES-buffered sucrose and then respin at 10,000 x g for another 15 min to yield the washed crude synaptosomal fraction (P2’).

5. layer P2’ fraction onto 4 ml of 1.2 M sucrose.

6. Centrifuge at 230,000 x g for 15 min in swinging bucket rotor. 38,000 rpm in SW 41 Ti, 44,000 rpm in SW50.1, 52,000 rpm in TLS-55.

7. Collect gradient interphase.

8. Dilute to ~7-8 ml with ice-cold HEPES-buffered sucrose (0.32 M sucrose, 4 mM HEPES pH 7.4).

9. Layer onto 4 ml of 0.8 M sucrose.

10. Centrifuge at 230,000 x g for 15 min.

11. Pellet contains pure synaptosomes.

12. For bioassays, wash and resuspend in appropriate incubation buffer:

For electroporation or functional release assays: (140 mM NaCl, 5 mM KCl, 5 mM NaHCO3, 1.2 mM Na2HPO4, 1 mM MgCl2, 20 mM HEPES pH 7.4, 10 mM dextrose (added fresh), ±1.8 mM CaCl2, and optional ATP regenerating system.

For digitonin permeabilization: 115 mM KOAc, 25 mM HEPES, pH 7.4, 5 mM NaOAc, 0.05 mM EGTA containing 0.025% digitonin, an ATP regenerating system, and 10 mM DTT. (see Meacham, Patterson, et al., Nature Cell Biology, 2001).

ATP regenerating system – 5 mM Mg-ATP, 80 mM creatine phosphate, 0.5 mg/ml creatine phosphokinase

12. Otherwise, can solubilize as desired, or subfractionate further into synaptosomal membranes and synaptic vescles by hypotonic lysis.

Even though some people use to isolate synaptosomes from frozen brain tissue, my experience lead me to believe that the quality of synaptosomal preparations from fresh brain is definitively higher than the quality obtained from frozen tissue.

This is particularly true when performing functional neurotransmitter release and/or uptake assays.

Good luck!

Dear Pascal, i think Bryens advice is the best advice to follow! Since hypertonic sucrose solutions such as 0.5 m sucrose or more, trigger synaptic vesicle exocytosis from presynaptic nerve terminals (by an unknown, Ca2+-independent mechanism) synaptosomes prepared by the "classical" protocols (like the one Jan mentioned), are not very suitable for your studies.

hi pascal we need to see the full protocol in order to help you. 5-HT uptake/release and synaptosomes is an every day task for me (the only things i do correctly ! ). philippe know me so feel free to send me your questions and protocol.

Hi, I'm a little bit late, but if you still need a protocol, I have one I could send you, at least for uptake.

Hi Johanna

Thanks, if you have a good protocol, I'm always interested. I now use crude synaptosome preparation, protocol given by Stephane Doly.

If you have time, can you send it to me?

thanks

I use one described by Gobbi et al, 2002. I standarized it for my laboratory, although it is in spanish. You'll find the article and the protocol (in spanish) attached. You can keep the pellet at -80°C. This protocol is really easy to perform, hope you'll find it useful for your research.

Regards

The protocol in spanish

Thanks! I'll try to remember my spanish...

Does anyone has an extraction protocol from primary cultured neurons?

Saber Hassan Did you manage to find a good working protocol for synaptosome extraction from primary neuron culture? I am currently looking for a procedure to follow.