Isolation of synaptosomes and glutamate release assay

Isolation of synaptosomes:

1. Prepare six Ficoll gradients (one gradient for each rat). From bottom to top: 4 mL 13%, 1 mL 9% and 4 mL 6% Ficoll solution
2. Decapitate six 5-6-week-old rats, excise the brains, remove the white matter as much as possible and homogenize the brains in 30 mL of ice-cold homogenization buffer in a glass-teflon homogenizer (add 30 µl of PMSF and 30 µl of pepstatin A stock solutions) – perform 9 strokes at 900 rpm.
3. Centrifuge the brain homogenate at 5000 rpm for 2 min using a SS34 rotor (Sorvall centrifuge) to pellet nuclei and cell debris.
4. Collect the supernatant S1 and discard the pellet P1. Centrifuge S1 at 11,000 rpm for 12 min (SS34 rotor, Sorvall).
5. Re-suspend the pellet P2, avoiding the dark brown part containing mitochondria) in 18 mL of homogenization buffer.
6. Load 3 mL each of the re-suspended pellet onto the Ficoll gradient using a pipette fitted with a tip of which the end is cut off to avoid any damage to synaptosomes by shearing forces.
7. Centrifuge the gradients using SW41 rotor (Beckmann centrifuge) at 22,500 rpm for 35 min.
8. Collect the synaptosomes, enriched in the 9-13 % Ficoll interface, using a Pasteur pipette and transfer them into a fresh SS34 tube for the next washing step.

9. Add 30 mL of homogenization buffer and centrifuge the suspension at 11,000 rom for 12 min, SS34 rotor. Re-suspend the resulting pellet P3 in 2.5 mL of homogenization buffer/rat brain, supplemented with PMSF and pepstatin A inhibitors. Keep the synaptosomes on ice until use.

    

Glutamate release assay:

1. Centrifuge 1 mg of synaptosomal protein at 8500 for 2 min, room temperature (the supernatant will be not cleared, to keep synaptosomes intact we do not increase the speed of centrifugation)
2. Very gently resuspend the pellet in 1 mL pre-warmed sodium buffer, again using a pipet with a blue tip of which the end is cut off
3. Incubate for5 min at 37°C in a water bath
4. Add 10 µl of NADP (final concentration: 1 mM) and 2.6 µl of CaCl₂ (final concentration: 1.3 mM) or 5 µl of EGTA (final concentration: 0.5 mM)
5. Transfer the sample in the glass cuvette using a cut blue tip and put the cuvette in the spectrofluorometer, set excitation wavelength to 340 nm and emission to 440 nm.
6. Record thesignal for3 min to obtain a stable baseline.
7. Add 50µl glutamate dehydrogenase (total 200 Units)
8. Record for 3 more min.
9. Stimulate synaptosomesby adding25 µl of 2 M KCl solution.
10. Recordthe responsefor another2-5 min.

Homogenization Buffer

320 mM sucrose, 5mM HEPES, pH 7.4 Sterile filtered and degassed. Store at 4°C

Freshly add protease inhibitors: Pepstatin (Final concentration 1µg/ml) and PMSF (Final concentration 200 mM)
 

Ficoll Gradients
6%, 9% and 13% (w/v) Ficoll in Homogenization buffer
From bottom to top: 4 mL 13%, 1 mL 9% and 4 mL 6% Ficoll

Sodium Buffer:
140 mM NaCl, 5 mM KCl, 20mM HEPES, 5 mM NaHCO₃, 1.2 mM Na₂HPO₄, 1 mM MgCl₂, 10 mM glucose, pH 7.4

NADP: 100 mM

CaCl2: 0.5 mM

EGTA: 100 mM

KCl: 2 M

Glutamate dehydrogenase from bovine liver: 200 Unit/reaction

Synaptosome isolation from rat brain (overview):

 

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