Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

All animal experimental procedures were approved by the University of Barcelona Committee on Animal Use and Care (CEEA), in compliance with the guidelines described in the Guide for the Care and Use of Laboratory Animals¹¹ and following the European Community, law 86/609/CCE, FELASA, and ARRIVE guidelines. Thus, mice are housed in standard cages, with ad libitum access to food and water, and are maintained under controlled standard conditions (12 h dark/light cycle starting at 7:30 AM, 22 °C temperature, and 66% humidity).

1. Obtaining Mouse Brain Synaptosomes Using a Discontinuous Sucrose Gradient

Note: This method was reported previously¹⁰.

1. Prepare fresh isolation buffer (IB), pH 7.4; 2 M sucrose; 1 M sucrose/0.1 mM CaCl₂; and 0.1 mM CaCl₂ (see Table 1). Chill the solutions on ice.
2. Sacrifice five mice by cervical dislocation. Decapitate and rapidly remove the brain of each animal. Dissect the brain region of interest (i.e., the hippocampus, 0.25-0.35 g of fresh tissue) and place it in a 5 mL Potter-Elvehjem glass tube on ice with 1 mL of IB.
3. Homogenize the tissue using a homogenizing stirrer (10 strokes at 700-900 rotations per min), preferentially in an ice bath to preclude sample warming.
4. Place the homogenized tissue (1 mL) into a 15 mL tube containing 6 mL of 2 M sucrose and 2.5 mL of 0.1 mM CaCl₂ at 4 °C. Mix slowly by inverting the tube.
   Note: The addition of calcium is essential to maintaining the adhesive interactions between organelles and thus to preserving the structure of the different "densities."
5. Place the solution in a 12 mL centrifuge tube and slowly add 2.5 mL of 1 M sucrose/0.1 mM CaCl₂ on top of each tube to form the sucrose gradient.
   Note: Label the position of the gradient interface on the centrifuge tube using a permanent marker.
6. Weigh and equilibrate the centrifuge tubes with IB solution within their corresponding steel supports and with their closing lids.
7. Centrifuge the tubes for 3 h at 100,000 x g and 4 °C using a swinging bucket rotor centrifuge. Completely fill the rotor with all the steel supports (even empty, if required).
8. Discard the top layer containing myelin. With a Pasteur pipette, collect the white ring between the 1.25-M and 1-M sucrose interphase corresponding to the synaptosome fraction.
9. Dilute the synaptosomes with 9X their volume using IB in a centrifuge tube.
10. Weigh and equilibrate the centrifuge tubes with IB solution within their corresponding steel supports and with their closing lids.
11. Centrifuge the tubes for 30 min at 15,000 x g and 4 °C using a swinging bucket rotor centrifuge.
12. Discard the supernatant and resuspend the pellet with 1.1 mL of IB solution. Collect 100 µL of this synaptosomal solution and centrifuge for 5 min at 11,000 x g and 4 ºC.
13. Resuspend the synaptosomal pellet in 5% sodium dodecyl sulfate (SDS) and store this sample at -20 ºC.
    Note: This sample will correspond to the total synaptosome fraction for further Western-blot analysis. The remaining synaptosomal fraction (~1 mL) can either be frozen down at -20 ºC until use or processed for subsequent subsynaptic fractionation. The synaptosomes or purified synapses represent between 1 and 2% of the total hippocampal volume¹².

2. Pre-, Post- and Extrasynaptic-isolation

1. Prepare 2x fresh solubilization buffer, pH 6.0; 1x solubilization buffer, pH 6.0; solubilization buffer, pH 8.0; and 0.1 mM CaCl₂ (see Table 1). Chill the solutions on ice.
   Note: The pH of these solutions should be accurately adjusted to achieve a good subsynaptic fractionation.
2. Slowly dilute the 1-mL resuspended synaptosomal fraction (see step 1.12) with 5 mL of 0.1 mM CaCl₂.
3. Add the same volume (5 mL) of ice-cold 2x solubilization buffer, pH 6.0, and incubate for 50 min on ice under high agitation in a beaker on ice.
   Note: While 1% of Triton X-100 at pH 6.0 solubilizes all plasma membrane proteins, it preserves proteins within the synaptic contacts or junctions (i.e., pre- and postsynaptic structures)⁴.
4. Place the solution in a centrifuge tube. Weigh and equilibrate the tubes with equivalent volumes of 0.1 mM CaCl₂ and 2x solubilization buffer solution, pH 6.0, within their corresponding steel supports and with their closing lids.
5. Centrifuge the tubes for 30 min at 40,000 x g and 4 °C using a swinging bucket rotor centrifuge. The resulting supernatant represents the extrasynaptic fraction, and the pellet corresponds to the synaptic junctions (i.e., pre- and postsynaptic fractions).
6. Concentrate the supernatant containing the extrasynaptic fraction to a final 200 µL volume using a 15 mL 10K filter tube centrifuged at 4,000 x g and 4 ºC.
7. Precipitate the resulting concentrated extrasynaptic fraction (200 µL) with 5 volumes (1 mL) of pre-chilled (-20 °C) acetone overnight at -20 °C.
8. The next day, centrifuge the extrasynaptic fraction for 30 min at 18,000 x g and -15 °C. After centrifugation, discard the acetone supernatant and dry the pellet to remove any trace of acetone.
9. Finally, resuspend the pellet containing the extrasynaptic proteins with 200 µL of 5% SDS. Sonicate the pellet, if required.
10. Without disrupting it, carefully wash the pellet containing the pre- and postsynaptic fractions with 2 mL of 1x solubilization buffer, pH 6.0. Discard the buffer.
11. Resuspend the pellet with 10 mL of 1x solubilization buffer, pH 8.0, using a glass Pasteur pipette.
    Note: While 1% of Triton X-100 at pH 8.0 solubilizes the presynaptic specialization, it preserves the insoluble postsynaptic density⁴.
12. Incubate the suspension for 50 min on ice under high agitation in a beaker on ice.
13. Place the solution in a centrifuge tube. Weigh and equilibrate the tubes with 1x solubilization buffer solution, pH 8.0, within their corresponding steel supports and with their closing lids.
14. Centrifuge the tubes for 30 min at 40,000 x g and 4 °C using a swinging bucket rotor centrifuge; the resulting supernatant corresponds to the presynaptic fraction and the pellet to the postsynaptic fraction.
15. Process the supernatant containing the presynaptic fraction, as described in steps 2.6-2.9.
16. Resuspend the pellet containing postsynaptic fraction with 200 µL of 5% SDS. Sonicate the pellet, if required.

3. Analyze the samples by immunoblot to validate the membrane fractionation

1. Determine the amount of protein in each fraction (i.e., the total, extra-, pre- and post-synaptic fractions) using the bicinchoninic acid protein assay.
2. Take 20 µg of protein from each fraction and dilute it to a final volume of 50 µL in SDS-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer. Boil for 5 min at 100 ºC.
3. Separate the proteins by SDS-PAGE electrophoresis 10%, with a 4% concentrating gel under reducing conditions. Electrophorese at constant voltage of 80 V until the dye enters the lower gel and then increase the voltage to 120 V.
4. Transfer the proteins to a PVDF membrane and block with IB blocking solution for 45 min at room temperature under continuous shaking.
5. Incubate the membrane overnight at 4 ºC with the indicated primary antibody (i.e., anti-SNAP-25, anti-PSD-95, anti-synaptophysin, or anti-GPR37) diluted in IB blocking solution under continuous shaking.
6. Wash the membrane three times (10 min each) with IB washing solution to eliminate unbound primary antibody.
7. Incubate with the indicated secondary antibody for 90 min in dark conditions at room temperature under continuous shaking.
8. Wash the membrane three times (10 min each) with IB washing solution to eliminate unbound secondary antibody.
9. Incubate the membrane with chemiluminescent substrate (prepare the mix under dark conditions and following the 1:1 proportion of solution A and B provided by the manufacturer).
10. Analyze the membrane in a chemiluminescent detection device.