Subcellular Fractionation for the Isolation of Synaptic Components from the Murine Brain

Synapses are the basic computational units of the brain. The presented method is a simple and valuable tool to study the synapses and their molecular processes and also to understand how they might be altered in various brain disorders. This technique involves a fractionation of synaptosome structures isolated from the brain.

This enables the researcher to capture effects that may be compartmentalized at the synapse. The distribution levels and activity of specific proteins can also be analyzed with subsynaptic resolution. This procedure takes around 11 hours for an individual researcher to complete if everything goes smoothly.

An individual who has never performed this technique may have difficulty with the length of this procedure as it will naturally take a little bit longer for someone who is unfamiliar with the steps. Because of the length of this experimental day, we highly recommend performing the dissections with a partner who can help you facilitate quick tissue collection to ensure that no samples set on ice for longer than is necessary. We also recommend preparing buffers and bench top materials the day prior to running this experiment so that everything is within easy reach when needed.

This is particularly true for the collection tubes. Multiple aliquots of a total of 11 subcellular fractions will be collected by the end of this procedure. So having these tubes clearly labeled ahead of time will make your day much easier.

To begin, insert fine scissors under the skin at the decapitation incision to a peri cranial depth and make a mid sagittal incision up to the intranasal suture to retract the scalp from the skull. Working from the occipital area toward each temporal aspect, trim the fascia and muscle to expose the external surface of the skull beyond each external acoustic meatus. Secure the scalp and rostral aspect of the skull with the non-dominant hand and with the other hand, insert fine scissors two millimeters into the caudal side of the foramen magnum where the spinal cord is visible exiting.

Make a midline incision until the scissors reach the internal surface of the intraparietal bone. Change the angle of the scissors so the blades run parallel to the dorsal surface of the skull. Continue advancing the midsagittal incision rostrally through the parietal and frontal bones using the sagittal and interfrontal sutures as a guide and terminate the incision just beyond the internasal suture.

Next, make a three millimeter perpendicular incision to the nasal bone, rostral to the internasal suture, by placing the scissors perpendicular to the skull with each blade positioned at a nasal premaxillary suture and making one even cut. While securing the rostral aspect, Use one side of a pair of textured forceps to gently lift the skull up from the brain and then lift it laterally and ventrally. Repeat along the midline as needed, then for the other hemisphere until the entire brain surface is exposed.

Using curved forceps or a fine spatula gently lift the rostral side of the brain. Cut the optic and cranial nerves to complete the excision from the skull. Homogenize the brains using a glass downs homogenizer placed on an ice bath in 12 up down passes at 500 rpm.

Pause briefly at each downstroke to ensure thorough homogenization of the tissue. Transfer the total brain homogenate to a 14 milliliter high speed round bottom centrifuge tube and spin it at 800 G for 10 minutes at four degrees Celsius to obtain the supernatant termed S1.transfer S1 to a new centrifuge tube, discard the pellet. Take two five microliter aliquots of S1 for the bicinchoninic acid assay and two 100 microliter aliquots of S1 for the western blot.

Spin S1 at 9, 000 G for 15 minutes at four degrees Celsius to obtain the synaptic somal supernatant, or S2, and crude synaptosome pellet or P2.Take two, 10 microliter aliquots of S2 for the bicinchoninic acid assay and two 500 microliter aliquots of S2 for the western blot. Discard the supernatant after obtaining the aliquots. after centrifuging at 9, 000 G for 15 minutes at four degrees Celsius, obtain the supernatant S2'and washed synaptosome, P2'Discard the supernatant and resuspend P2'in three milliliters of buffer A.Avoid resuspending the dark red portion at the bottom of the pellet, which mainly contains mitochondria.

Take two 20 microliter aliquots of P2'for the bicinchoninic acid assay and two 100 microliter aliquots of P2'for the western blot. For the hypotonic lysis of washed synaptosome add nine volumes of chilled buffer B to resuspend P2'and homogenize the synaptosome in a glass down homogenizer. Transfer the samples to 50 milliliter capped conical centrifuge tubes and rotate them on a tube revolver in a four degree Celsius cold room for 15 minutes.

Centrifuge the lysed P2'at 25, 000 G for 20 minutes at four degrees Celsius to obtain the lysis supernatant or LS1, and the lysis pellet containing synaptosomal membranes, or LP1. Take two, 50 microliter aliquots of LS1 for the bicinchoninic acid assay and two 400 microliter aliquots of LS1 for the western blot. Transfer LS1 into a capped centrifuge tube for ultra centrifugation.

Centrifuge LS1 in a fixed angle ultra centrifuge rotor at 100, 000 G for 60 minutes at four degrees Celsius to obtain synaptic cytosol supernatant, or LS2, and synaptic vesicle pellet, or LP2. Resuspend LP2 in 500 microliters of buffer A and using a 23 gauge needle and a one milliliter syringe sheer LP two with gentle trituration. Take two, 10 microliter aliquots of LP2 for the bicinchoninic acid assay and two, 250 microliter aliquots of LP2 for the western blot.

Transfer around 30 milliliters of LS2 to centrifugal filter units with a 10 miliDalton cutoff and concentrate the LS2 to approximately 0.5 milliliters by spinning at 5, 000 G for up to one hour at four degrees Celsius. Take two, 10 microliter aliquots of concentrated LS2 for the bicinchoninic assay, and two, 250 microliter aliquots of concentrated LS2 for the western blot. Resuspend LP1 in one milliliter of buffer B, And take two, 10 microliter aliquots of LP1 for the bicinchoninic acid assay and two 50 microliter aliquots of LP1 for the western blot.

Adjust the remaining LP1 to a final volume of 7.5 milliliters, and a final sucrose concentration of 1.1 molar with buffer B and buffer C.Then transfer 7.5 milliliters of the resuspended LP1 into a 14 milliliter ultra centrifuge tube. Carefully overlay the LP1 with 3.75 milliliters of buffer D and mark the top of the solution with a pen. Then overlay with around 1.25 milliliters of buffer A.And similarly mark the top of the solution with a pen.

Balance the tubes for ultra centrifugation by weight, not volume, with the drop-wise addition of buffer A to within 10 milligrams. Centrifuge at 48, 000 G for 2.5 hours at four degrees Celsius in a swinging bucket ultra centrifuge rotor and acquire images of the intact gradients to document the distinctness of each sucrose interface and the success of fractionation. Carefully remove the superficial layer of 320 millimolar sucrose and recover the myelin fraction at the 320 millimolar, 855 millimolar sucrose interface in an 800 microliter volume.

Pipette up from the tubes wall in a circular manner to ensure the complete fraction is collected. Then recover the SPM fraction at the 855 millimolar, 1.1 millimolar sucrose interface in a 1000 microliter volume. Carefully aspirate off the remaining sucrose and recover the mitochondrial pellet by resuspending in 200 microliters of buffer B.Dilute the SPM fraction with two volumes of buffer B, and then centrifuge in a fixed angle rotor in a 3.5 milliliter centrifuge tube.

Discard the supernatant and resuspend the SPM pellet in buffer A for a final volume of 250 microliters. Take two, five microliter aliquots of SPM for the bicinchoninic acid assay and divide the remaining SPM in half for the western blot. Electron microscopy images of synaptosome are shown in this figure.

A synaptosome containing synaptic vesicles both pre and post synaptic components and synaptic vesicles in a mitochondrion are shown here. Immunoblot analysis of subcellular fractions was performed to assess markers of fraction purity. When compared to the initial whole brain homogenate, the analysis revealed the enrichment of n-cadherin in the synaptic plasma membrane fraction, alpha synuclein in the synaptic cytosol, synaptophysin in the synaptic vesicle fraction, and myelin basic protein in the myelin fraction.

Once fraction purity has been established, quantitative immunoblotting can be used to determine the localization of proteins of interest, or query differences in protein distribution between genotypes or treatments. When attempting this procedure, make sure to use detergent free glassware so that intact synaptosome can be collected. Also, be careful while preparing sucrose gradients to ensure that you obtain clear interfaces.

This will allow you to successfully isolate the synaptic plasma membrane fraction. Various structural and functional analysis can be performed to assist the quality of synaptic fractions such as electron microscopy, immunoblotting, proteomics, and other molecular methods. Neurotransmitter release or enzymatic assays can also be performed to assist the metabolic viability of synaptosome.

The isolation of synaptosome structures was a paradigm shifting technique for the analysis of synapse function and composition. As we see early synaptic dysfunction in neurodegeneration the careful dissection of molecular changes at the synapse level will help us explore the molecular mechanisms of these disorders.