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

The overall goal of this procedure is to isolate proteins belonging to different synaptic compartments from mice brain. Describing the distribution of kinetic transmissioncomponents is essential to understand synaptic function both in normal and pathological conditions. Accumulating evidence show that in vivo synapses are extremely dynamic and can undergo dramatic structural changes open sustaining synaptic transmission.

Synapses have been classically studied by means of electron microscopy. This membrane fractionation method is based on combination of laser obliteration using mild detergents under different pH conditions followed by split second centrifugation. In brief, mouse brain is removed and its hippocampus is carefully dissected in a nice cold dish seated on crushed ice.

The hippocampi are collected into a Potter-Elvehjem glass tube containing ice cold isolation buffer. Subsequently, the collected tissue is homogenized using a homogenizing stirrer. Next the homogenized tissue is mixed with a sucrose calcium solution.

Then the tissue extract is overlaid with a concentrated sucrose calcium solution. The samples are then centrifuged. The membrane fraction containing synaptosomes is easily recognized as a dense ring floating within the sucrose solution's interface.

Subsequently the isolation of the different sub-synaptic compartments, namely extra, pre, and post synaptic fractions is achieved by combining light solubilization using mild detergents upon differentiation pH condition. Finally, the protein enrichment at the different compartments is validated by immunoblot analysis. The day of the experiment prepare fresh solutions and chill them on ice.

Place the brain in a Petri dish seating on crushed ice and containing 10 milliliters of ice cold isolation buffer. Carefully dissect the hippocampus with the help of a pair of forceps. Transfer the hippocampi into a five milliliters Potter-Elvehjem glass tube containing one milliliter of ice-cold isolation buffer.

And then homogenize using a homogenizing stirrer. Transfer the homogenized tissue into a 15 milliliters Falcon tube containing the sucrose and calcium solution and mix it slowly by inverting the tube. Place the solution in an ultra-clear centrifuge tube and add on top of each tube 2.5 milliliters of sucrose containing calcium chloride.

And calibrate the tubes with isolation buffer solution and centrifuge for three hours at 100, 000 G at four degrees in an ultra centrifuge. Carefully remove the tube from the centrifuge steel support and identify the dense ring within the sucrose interface which contains the synaptosomes. Discard the top mailing layer with the help of a blue tip.

With a Pasteur pipette, collect the dense ring containing the synaptosomes fractions and transfer into an ultra-clear centrifuge tube and keep on crushed ice. Dilute the synaptosomes with nine volume of ice cold isolation buffer and centrifuge for 30 minutes at 15, 000 G at four degrees. After centrifugation, remove the tube.

Discard the supernatant. A pellet containing the synaptosomes should be seen. Resuspend the pellet with 1.1 milliliters of isolation buffer and keep on crushed ice for the next step.

Make fresh solutions with the corresponding pH adjusted at four degrees. Add slowly one milliliter of the resuspended synaptosomal pellet into five milliliters of calcium chloride in a beaker on ice under continuous agitation. Subsequently at five milliliters of concentrated solubilization buffer pH 6.

After 50 minutes on ice, transfer the solution into an ultra-clear centrifuge tube and centrifuge at 40, 000 G for 30 minutes at four degrees. After centrifugation, remove the tube. A pellet containing the pre and post-synaptic fraction should be observed.

The supernatant containing the extra synaptic fraction is collected in a 15 milliliters concentrating tube. Carefully wash the pellet with the two milliliters solubilization buffer pH six. Avoid disrupting the pellet and discard the buffer.

At 10 milliliters of solubilizing buffer pH eight to the pellet, and titrate with the aid of a Pasteur pipette. Incubate a beaker on ice under continuous agitation during 50 minutes. Transfer the solution into an ultra-clear centrifuge tube and centrifuge at 40, 000 G for 30 minutes at four degrees.

After centrifugation, remove the tube. A pellet containing the post-synaptic fraction should be observed. The supernatant containing the pre-synaptic fraction is collected in a 15 milliliters concentrating tube.

The pellet containing the post-synaptic fraction is resuspended with 200 microliters of STS solution. Sonicate the resuspended pellet. Assess the amount of protein in each fraction by means of a protein determination assay.

Take 20 micrograms of protein from each fraction and treat with STS Polyacrylamide Gel Electrophoresis sample buffer. Separate proteins by STS Polyacrylamide Gel Electrophoresis and analyze by immunoblot. GPR37 is another fancy protein global receptor mostly in rodent brain.

Implicated in different pathologies such as Parkinson's disease or major depression, its function remains abnormal. Studying the presence of this receptor in synapses can help decipher its role in the central nervous system. Synaptosomes from wild type and GPR37 knockout mice were isolated and subsequently fractionated.

The purity of each sub-synaptic compartment was warrantied by the segregation of the respective synaptic markers. The extra synaptic vesicular marker synaptophysin was enriched in this extra synaptic fraction. The BSD 95 protein, a classical post-synaptic density marker was enriched in the corresponding post synaptic fraction.

Finally, the purity of pre synaptic fraction was validated by the presence of SNAP 25 a pre synaptic active zone marker. Interestingly while GPR 37 immuno reactivity was more abundant in the extra synaptic fraction, it also showed a significant preference for the post synaptic over the pre-synaptic fraction. Assessing the sub synaptic protein composition is an important challenge in neuroscience.

However, this is not an easy task since it is highly regulated by protein protein interaction ribbons. Here we present a brain membrane fractionation protocol that represent a robust procedure to select proteins belonging to different synaptic compartments. In other words, it is described of your chemical technology to carry out protein enrichment from pre synaptic, post synaptic, and extra synaptic compartments.

Goodbye and good luck with your fractionation experiments.