Name: Preparation of Synaptoneurosomes from Mouse Cortex using a Discontinuous Percoll-Sucrose Density Gradient (Video) | JoVE Journal
Uploaded: 2011-09-17T14:00:00.000+00:00
Duration: 8 min 30 s
Description: Learn to prepare translationally active synaptoneurosomes from mouse cortex using a discontinuous Percoll-sucrose density gradient. This method minimizes mechanical damage and enables reliable study of synaptic protein synthesis.

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5 minutes

We would like to thank B. K. August from the University of Wisconsin-Madison Electron Microscope Facility for the electron microscopy. This work was supported by NIH grants R01-DA026067 and P30-HD03352 (to J.S.M.).

1. Preparations1-4
<ol>
 <li>Prepare 500 mL of gradient medium (GM) buffer by mixing 50 mL of a 2.5 M sucrose slurry, 2.5 mL of a 1M Tris-HCl, pH7.5 stock, and 0.1 mL of a 0.5 m EDTA, pH 8.0 stock with milli-Q water to volume. Filter sterilize the solution, aliquot and store frozen.</li>
 <li>Prepare a 1000x stock of tetrodotoxin (TTX) by making a 1 mM solution in milli-Q H2O. Aliquots of TTX can be stored at -20°C.</li>
 <li>Prepare 10x Stimulation Buffer by making a solution of 100 mM Tris (pH 7.5), 5 mM Na2HPO4, 4 mM KH2PO4, 40 mM NaHCO3, and 800 mM NaCl in milli-Q water. Stock can be stored at 4°C.</li>
 <li>Pre-cool centrifuges to 4°C.</li>
 <li>Prepare a stock solution of Isosmotic Percoll (SIP) by adding 9 volumes of Percoll (18 mL) to 1 volume of 2.5 M sucrose (2 mL) in milli-Q water and mix well.</li>
 <li>Prepare the 3, 10, 15, and 23% gradient layers by adding the respective amounts of SIP to GM buffer and mixing each:</li>
</ol>
<img alt="Gradient table with GM buffer and SIP volumes for chromatography experiment data analysis." src="/files/ftp_upload/3196/3196table0.jpg" data-altupdatedat="1788235205000" data-alt="Table 0">
<ol start="7">
 <li>Pour gradient layers by pipetting 2 ml each of the 23, 15, 10, and 3% isosmotic Percoll solutions into the Beckman centrifuge tubes with caps using a P1000 Gilson Pipetman. The interface between the layers should be clearly discernable with no mixing of the layers. Store the gradients at 4°C for at least 20 min before use. The gradients can be stored for up to 24 hours although same day use is recommended. [Inexperienced lab personnel may practice preparing gradients by adding blue dye to the isomotic Percoll solutions for visualization of the layer interfaces.]</li>
</ol>
2. Mouse dissection 1
<ol>
 <li>Ensure that all mouse husbandry and euthanasia procedures are performed in accordance with NIH and University guidelines. Euthanize pups (age P13 to P21) by carbon dioxide asphyxiation or by a method approved of in the animal protocol.</li>
 <li>Pin the mouse to a dissection board and spray the back of the neck and head with ethanol. Using a sharp scissors cut through the spinal cord at the base of the skull, remove the skin from the top of the skull, cut the skull laterally between the parietal bone and the interparietal bone or between the cerebrum and the cortex regions. Then cut the skull from the base to the nose (along the sagital suture), carefully remove the soft parietal bone by pulling each hemisphere to the side and remove the cortex with a spatula. Insert the spatula into the brain above the cerebellum and then scoop out the cortex and place it in ice-cold GM buffer. Proceed immediately to the homogenization step. Do not let the cortices sit in ice-cold buffer for very long as this will adversely affect the formation of SNs.</li>
</ol>
3. Preparation of homogenate and SNs 1-4
<ol>
 <li>Rinse the cortices in ice-cold GM buffer and transfer two cortices to a glass Dounce homogenizer containing 5 mL of cold GM buffer.</li>
 <li>Gently homogenized the cortices with 5-10 strokes of the loose pestle (pestle "A"), followed by 5-10 strokes of the tight pestle (pestle "B"). Strokes should be rapid but slow enough not to cause air bubbles. A picture of the homogenate after homogenization is in Scheme 1. The number of strokes will vary dependent on the individual homogenizer and accompanying pestles. When finished the gradients should give a strong SN band, if not, adjust the number of strokes.</li>
 <li>Transfer the homogenate to a 15 mL conical and centrifuge at 1000xg for 10 min at 4°C in a swinging bucket rotor (Allegra 6KR centrifuge) to pellet cellular debris and nuclei. A Picture of the spun homogenate is in Scheme 1.</li>
 <li>Layer 2 mL of the supernatant per Percoll-sucrose gradient, cap the tubes, and centrifuge at 32,500xg for 5 min at 4°C in a fixed-angle rotor (Beckman J2-21 centrifuge, JA-17 rotor) using appropriate adapters (See Reagents and Equipment Table).</li>
 <li>Pipette off and discard the solution above the SN band using a glass Pasteur pipette. Pipette off the SN band at the 15/23% interface, transfer to a conical and store on ice. Each gradient or one cortex will produce 0.9-1.1 mL of SN.</li>
 <li>Adjust the salt concentration of the SNs by adding one-tenth volume of 10x stimulation buffer, add 1000x CaCl2 to a final concentration of 12 nM, and add 1 mM TTX stock to a final concentration of 1 μM to suppress nonspecific excitation. (Addition of CaCl2 is optional if it will interfere with downstream SN applications).</li>
 <li>Determine the protein concentration of the SNs using the Micro BCA Protein Assay Kit. (The Bradford protein assay is not recommended because of inference from the Percoll.)</li>
 <li>SNs can be used directly and quickly for protein translation studies. For other applications SN lysate may be cleaned up or concentrated using the Pierce SDS-PAGE Sample Prep Kit.</li>
</ol>
4. Representative results:
When mouse cortex was homogenized and separated on discontinuous Percoll-sucrose gradients (Scheme 1), 6 bands or fractions were seen (Figure 1). It was previously shown for rat cerebral cortex3,5 that the constituents of the high molecular weight bands were broken membrane and myelin and that the pelleted material contained mitochondria or organelles (Table 1). The 23%/15% interface (Band 5) contained the enriched SNs.
The band at the 23%/15% interface, containing intact SNs, was removed and examined by electron microscopy (EM) as described previously.2,6 EM showed the presence of intact synaptic vesicles and the preservation of presynaptic and postsynaptic elements (Figure 2). Isolation of pre- and postsynaptic compartments is important for examining signaling and protein synthesis, which occurs in both compartments.7-13 The Percoll-sucrose gradient is a crude purification so we saw minor cellular, nuclear and organelle contamination in the SN fraction but the overall separation was good.
When examined by Western blot, the Percoll fractions contained the expected protein markers with an increase in synaptic purity in the SN band (Figure 3, Table 2). Synaptic and neuronal markers were maintained in the enriched SN band (Band 5) but the abundance of other markers was significantly reduced. In particular, the presence of GFAP, a marker for astrocyte and neoplastic cells of glial origin, and Lamin-B1, a nuclear marker, were negligible, while structural and synaptic markers were maintained or enhanced. In addition, there was retention of markers for organelles, such as the mitochondria and Golgi, which play a role in protein synthesis.
Typical yields for the SN band were 250-500 ng of protein per μL, as determined by BCA protein assay. The activity of the SNs was determined by the amount of de novo protein synthesis present as monitored by [35S]-Met incorporation (Figure 4).2 Basal translational activity was increased 1.56 fold when SNs were stimulated with 50 μM glutamate/10 μM glycine (Glu) or 5.12 fold when activated with the Pin1 inhibitor juglone. Pin1 inhibition is known to result in increased protein synthesis.2 To confirm that the increase in [35S]-Met incorporation was due to de novo protein synthesis, we added 40 μM anisomycin, a protein synthesis inhibitor, and saw a marked decrease in incorporation in the presence and absence of Glu when compared to basal levels. Also, upon the addition of 2% Triton-X 100, which disrupts the integrity of the SNs, basal translation was decreased by 75%.2 In addition, when examining the bands (B4-B6) that showed the most synaptic marker enrichment, the SNs or Band 5 had the greatest activity for de novo protein synthesis (Figure 5). Thus, the discontinuous Percoll-sucrose gradients quickly produce a highly active and enriched SN band that can be used to study protein translation.
The discontinuous Percoll-sucrose gradient procedure is more advantageous than other methods because mechanical damage is caused by harsher conditions. When compared to the filtration method, in which cleared cortical homogenate is passed through a series of filters that decrease in size,3,14-15 the Percoll method forms more SNs with greater activity. As seen in Figure 6, when an aliquot of SNs prepared by the filtration method was passed over a Percoll-sucrose gradient there are far less SNs at the 15/23% interface and a greater amount of broken membranes. When de novo protein synthesis was measured via S35-met incorporation, the SNs prepared by the filtration method were far less active (Figure 7). To maximize translational activity we use mice that are between age P14 and P18. SN can be prepared from adult mice, but we observed significantly increased translational activity with SN prepared from juvenile mice (Figure 8).
<img alt="Cell component distribution, table, showing bands with membranes, myelin, SNs, organelles." src="/files/ftp_upload/3196/3196table1.jpg" data-altupdatedat="1788235205000" data-alt="Table 1"> 
Table 1. Constituents of the bands from the discontinuous Percoll-gradient fractionations of homogenized mouse cortex.3,5
<img alt="Protein marker distribution chart; lab results showing protein presence in different bands." src="/files/ftp_upload/3196/3196table2.jpg" data-altupdatedat="1788235205000" data-alt="Table 2"> 
Table 2. Summary of Western bolt analysis of discontinuous Percoll-sucrose gradient band constituents. Relative protein concentrations for each of the band fractions isolated using a discontinuous Percoll-sucrose gradient were determined by Western blot. No protein present (-), 0≥0.2 (+), 0.2≥0.8 (++), or &gt;0.8 (+++) fold greater than protein present in the supernatant (S, centrifuged cortex homogenate), representative of n=3.
<img alt="Cortex dissection and centrifugation diagram; shows homogenization, Percoll gradients, SN Band." src="/files/ftp_upload/3196/3196scheme1.jpg" data-altupdatedat="1788235205000" data-alt="Scheme 1"> 
Scheme 1: Schematic of a SN preparation using a discontinuous Percoll-sucrose density gradient. Mouse brain cortices were dissected, homogenized, and centrifuged at low speed to remove non-homogenized tissue, cells, whole organelles such as nuclei and membranes. The discontinuous gradient should have clearly defined layers. The picture inset shows an example of the layers, which have been dyed blue for illustrative purposes only in order to visualize the layers. The supernatant is layered on top of a discontinuous Percoll-sucrose gradient and centrifuged at medium speed. The SN band is then retrieved from the gradient and is ready to use.
<img alt="Density gradient centrifugation diagram; protein separation, gradient percentages, band identification." src="/files/ftp_upload/3196/3196fig1.jpg" data-altupdatedat="1788235205000" data-alt="Figure 1"> 
Figure 1. Homogenate fractionation on a discontinuous Percoll-sucrose gradient. Mouse cortex was homogenized and separated on a discontinuous Percoll-sucrose gradient giving rise to 6 bands or fractions. The purified, active SNs (*) were found in Band 5.
<img alt="Electron microscopy image; cell ultrastructure analysis, organelles highlighted with arrows." src="/files/ftp_upload/3196/3196fig2.jpg" data-altupdatedat="1788235205000" data-alt="Figure 2"> 
Figure 2. SN preparations give a heterogeneous mix of SNs that retain presynaptic and postsynaptic elements. An electron micrograph of an SN preparation prepared from C57BL/6 mice (age P13 to P21) was performed as previously described and shows SNs with preserved presynaptic and postsynaptic elements.2,6 Red arrows point to post-synaptic densities. Scale bar is 1 μm.
<img alt="Western blot diagram; protein expression analysis of β-Actin, GFAP, GP73 across bands 1-6, S." src="/files/ftp_upload/3196/3196fig3.jpg" data-altupdatedat="1788235205000" data-alt="Figure 3"> 
Figure 3. Western blot analyses of SN preparations showed that synaptic and neuronal markers were maintained in the purified SNs (Band 5) but the abundance of other markers was significantly reduced. Immunoblots of supernatant (S, centrifuged cortical homogenate) and Bands 1-6 of a discontinuous Percoll-sucrose gradient were probed for β-Actin (structural, loading control), GFAP (astrocytic), GP73 (Golgi), HSC70 (cytoplasmic and nuclear), Lamin-B1 (nuclear), Prohibitin (mitochondrial), PSD95 (postsynaptic density), SNAP25 (synaptic), Synaptophysin (synaptic) and β3-Tubulin (neuron-specific). The bands were visualized using a Storm 860 Phosphorimager, representative of n=3.
 
<img alt="SDS-PAGE protein electrophoresis and S35-Met incorporation bar chart for drug effect analysis." src="/files/ftp_upload/3196/3196fig4.jpg" data-altupdatedat="1788235205000" data-alt="Figure 4"> 
Figure 4. SNs are active and exhibit de novo protein synthesis via [35S]-Met incorporation. SNs were pre-equilibrated to 37°C for 10 min. Then the SNs were untreated or pretreated for 15 min with 40 μM anisomycin or 1 μM juglone at 37°C followed by addition of [35S]-Met, 20 μl Express Pro Label Mix S35 Easy Tag, plus or minus Glu at 37°C for 30 min. The samples were prepared using the Pierce SDS-PAGE Sample Prep Kit, run on a 15% polyacrylamide gel, dried and quantitated using a Molecular Dynamics Storm 860 phosphorimager, representative of n=3, ±SEM.
<img alt="Protein electrophoresis and S35-Met incorporation graph; gel bands, bar chart comparison, Glu context." src="/files/ftp_upload/3196/3196fig5.jpg" data-altupdatedat="1788235205000" data-alt="Figure 5"> 
Figure 5. Band 5 from the discontinuous Percoll-sucrose gradient contained the highest levels of de novo protein synthesis. Bands 4, 5 and 6 and the supernatant (S, centrifuged cortical homogenate) were pre-equilibrated to 37°C for 10 min. Then, [35S]-Met, Express Pro Label Mix S35 Easy Tag, was added, plus or minus Glu at 37°C for 30 min. The samples were prepared using the Pierce SDS-PAGE Sample Prep Kit, run on a 15% polyacrylamide gel, dried and quantitated using a Molecular Dynamics Storm 860 phosphorimager, representative of n=3, ±SEM.
<img alt="Protein purification process in ultracentrifuge tubes; sediment and supernatant separation." src="/files/ftp_upload/3196/3196fig6.jpg" data-altupdatedat="1788235205000" data-alt="Figure 6"> 
Figure 6. SNs prepared from a filtration method contain more broken membranes and less whole SNs than SNs prepared from the discontinuous Percoll-sucrose gradient method. SNs were prepared by passing homogenized cortex through a series of filters with decreasing pore size as described previously.3,14-15 An aliquot of the SNs prepared by the filtration method was centrifuged over a discontinuous Percoll-sucrose gradient and compared to an equivalent amount of material using the discontinuous Percoll-sucrose gradient method.
<img alt="SDS-PAGE gel and bar graph showing protein analysis; filtration vs. percoll results in experiment." src="/files/ftp_upload/3196/3196fig7.jpg" data-altupdatedat="1788235205000" data-alt="Figure 7"> 
Figure 7. SNs prepared from a filtration method contain less de novo protein synthesis activity than SNs prepared from the discontinuous Percoll-sucrose gradient method. SNs were prepared by passing homogenized cortex through a series of filters with decreasing pore size as described previously,3,14-15 and by the discontinuous Percoll-sucrose gradient methods. SNs from both preparations were pre-equilibrated to 37°C for 10 min. Then, [35S]-Met, Express Pro Label Mix S35 Easy Tag, was added, plus or minus Glu at 37°C for 30 min. The samples were prepared using the Pierce SDS-PAGE Sample Prep Kit, run on a 15% polyacrylamide gel, dried and quantitated using a Molecular Dynamics Storm 860 phosphorimager, representative of n=3, ±SEM.
<img alt="Polyacrylamide gel electrophoresis and bar chart; protein synthesis analysis, S35-Met incorporation." src="/files/ftp_upload/3196/3196fig8.jpg" data-altupdatedat="1788235205000" data-alt="Figure 8"> 
Figure 8. SNs from younger mice (P14) have greater translational activity than older mice (P85). SNs were prepared from different aged mice using the discontinuous Percoll-sucrose gradient method, preequilibrated at 37°C for 10 min, treated or untreated with Glu in the presence of [35S]-Met (Express Pro Label Mix S35 Easy Tag) for 30 min, snap frozen, and later cleaned up with the Pierce SDS-PAGE Sample Prep Kit. The SNs were then run on a 12% SDS-polyacrylamide gel, dried and imaged on a Molecular Dynamics Storm 860 phosphorimager, representative of n=3, ±SEM. SNs from P14 mice were at least 3-fold more active than P85 mice.

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<li>Gray, E. G., Whittaker, V. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+isolation+of+nerve+endings+from+brain%3A+An+electron+microscopic+study+of+cell+fragments+derived+by+homogenization+and+centrifugation%5BTitle%5D)+AND+%22J.+Anat%22%5BJournal%5D)">The isolation of nerve endings from brain: An electron microscopic study of cell fragments derived by homogenization and centrifugation.</a> J. Anat. 96, 79-88 (1962).</li>
<li>Gylys, K. H., Fein, J. A., Yang, F., Cole, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enrichment+of+Presynaptic+and+Postsynaptic+Markers+by+Size-Based+Gating+Analysis+of+Synaptosome+Preparations+From+Rat+and+Human+Cortex%5BTitle%5D)+AND+%22Cytometry+A%22%5BJournal%5D)">Enrichment of Presynaptic and Postsynaptic Markers by Size-Based Gating Analysis of Synaptosome Preparations From Rat and Human Cortex.</a> Cytometry A. 60, 90-96 (2004).</li>
<li>Hajós, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+improved+method+for+the+preparation+of+synaptosomal+fractions+in+high+purity%5BTitle%5D)+AND+%22Brain+Res%22%5BJournal%5D)">An improved method for the preparation of synaptosomal fractions in high purity.</a> Brain Res. 93, 485-489 (1975).</li>
<li>Bai, F., Witzmann, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Synaptosome+Proteomics%5BTitle%5D)+AND+%22Subcell.+Biochem%22%5BJournal%5D)">Synaptosome Proteomics.</a> Subcell. Biochem. 43, 77-98 (2007).</li>
<li>Rao, A., Steward, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evidence+that+protein+constituents+of+postsynaptic+membrane+specializations+are+locally+synthesized%3A+analysis+of+proteins+synthesized+within+synaptosomes%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Evidence that protein constituents of postsynaptic membrane specializations are locally synthesized: analysis of proteins synthesized within synaptosomes.</a> J Neurosci. 11, 2881-2895 (1991).</li>
<li>Pertoft, H., Laurent, T. C. Isopycnic separation of cells and cell organelles by centrifugation and modified colloidal silica gradients. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aPertoft%2C+H+%22Methods+of+Cell+Separation%22">Methods of Cell Separation</a>. Catsimpoolas, N. , Plenum Press. New York. 25-65 (1977).</li>
<li>Ramarao, C. S., Acharya, S. R., Krishnan, K. S., Kenkare, U. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High+affinity+uptake+of+L-glutamate+and+%26gamma%3B-aminobutyric+acid+in+Drosophila+melanogaster%5BTitle%5D)+AND+%22J.+Biosci%22%5BJournal%5D)">High affinity uptake of L-glutamate and &gamma;-aminobutyric acid in Drosophila melanogaster.</a> J. Biosci. 11, 119-135 (1987).</li>
<li>Munteanu, L. S., Dinu, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fractionation+of+granulocytes+from+whole+human+blood+by+centrifugation.+Practical+hints%5BTitle%5D)+AND+%22Romanian+J.+Biophys%22%5BJournal%5D)">Fractionation of granulocytes from whole human blood by centrifugation. Practical hints.</a> Romanian J. Biophys. 14, 53-58 (2004).</li>
<li>Cell separation composition, kit and method. US patent. , 019713 (1997).</li>
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No conflicts of interest declared.

The discontinuous Percoll-sucrose gradient preparation described herein is a quick, reliable method to isolate active SNs, which can be used in a variety of synaptic transmission experiments. This gradient method, which is based on the method developed by Dunkley et al.,3,4 is a subcellular brain fractionation procedure that isolates both pre- and postsynaptic membrane-derived vesicles that are associated with one another. Without further purification these SNs are compatible with a variety of molecular biolog...

<table><tbody><tr><td> Name of the reagent</td><td> Company</td><td> Catalogue number</td><td> Comments (optional)</td></tr><tr><td>Micro BCA Protein Assay Kit</td><td>Pierce</td><td>23235</td><td>&amp;nbsp;</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Fisher</td><td>C79-500</td><td>&amp;nbsp;</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; gas</td><td>Airgas (UW-MDS)</td><td>CD 50</td><td>&amp;nbsp;</td></tr><tr><td>EDTA</td><td>RPI</td><td>E57020</td><td>&amp;nbsp;</td></tr><tr><td>EtOH</td><td>Fisher</td><td>A407SK-4</td><td>&amp;nbsp;</td></tr><tr><td>HCl</td><td>Fisher</td><td>A142-212</td><td>&amp;nbsp;</td></tr><tr><td>Percoll</td><td>GE Healthcare</td><td>17-0891-01</td><td>&amp;nbsp;</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher</td><td>P285-500</td><td>&amp;nbsp;</td></tr><tr><td>PierceSDS-PAGE Sample Prep Kit</td><td>Pierce</td><td>89888</td><td>&amp;nbsp;</td></tr><tr><td>NaCl</td><td>RPI</td><td>S23020</td><td>&amp;nbsp;</td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Fisher</td><td>BP328-500</td><td>&amp;nbsp;</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;PHO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher</td><td>S381-500</td><td>&amp;nbsp;</td></tr><tr><td>Sucrose</td><td>RPI</td><td>S24060</td><td>&amp;nbsp;</td></tr><tr><td>Tris Base</td><td>RPI</td><td>T60040</td><td>&amp;nbsp;</td></tr><tr><td>Tetrodotoxin</td><td>Sigma</td><td>T5651</td><td>&amp;nbsp;</td></tr><tr><td>Express Pro Label Mix S35 Easy Tag</td><td>Perkin Elmer</td><td>NEG772</td><td>&amp;nbsp;</td></tr><tr><td>&amp;nbsp;</td><td>&amp;nbsp;</td><td>&amp;nbsp;</td><td>&amp;nbsp;</td></tr><tr><td> Equipment</td><td> Company</td><td> Catalogue number</td><td> Comments (optional)</td></tr><tr><td>Dissection tools</td><td>&amp;nbsp;</td><td>&amp;nbsp;</td><td>&amp;nbsp;</td></tr><tr><td>Dounce homogenizer, 7 mL (comes with two glass pestles labled &quot; A&quot; and &quot;B&quot;)</td><td>Wheaton</td><td>&amp;nbsp;</td><td>&amp;nbsp;</td></tr><tr><td>P1000 Gilson Pipetman</td><td>Gilson</td><td>F123602</td><td>&amp;nbsp;</td></tr><tr><td>Allegra 6KR Centrifuge</td><td>Beckman Coulter</td><td>366830</td><td>&amp;nbsp;</td></tr><tr><td>GH 3.8 Rotor, Swinging bucket rotor</td><td>Beckman Coulter</td><td>360581</td><td>&amp;nbsp;</td></tr><tr><td>Beckman J2-21 Centrifuge</td><td>Beckman</td><td>&amp;nbsp;</td><td>&amp;nbsp;</td></tr><tr><td>Beckman tubes with caps</td><td>Beckman</td><td>355672</td><td>&amp;nbsp;</td></tr><tr><td>White walled adapters</td><td>Beckman</td><td>342327</td><td>&amp;nbsp;</td></tr><tr><td>Blue walled adapters</td><td>Beckman</td><td>&amp;nbsp;</td><td>&amp;nbsp;</td></tr><tr><td>JA-17 Rotor, Fixed-angle rotor</td><td>Beckman</td><td>369691</td><td>&amp;nbsp;</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td colspan="4">&lt;p class=&quot;jove_step&quot;&gt;Table of antibodies used for western blots:&lt;/p&gt;</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> Name of Antibody</td><td> Company</td><td> Catalogue number</td><td> Host Species</td></tr><tr><td>&amp;beta;-Actin</td><td>Sigma</td><td>A5441</td><td>mouse</td></tr><tr><td>GFAP</td><td>Santa Cruz</td><td>sc-65343</td><td>mouse</td></tr><tr><td>GP73</td><td>Santa Cruz</td><td>Sc-134509</td><td>rabbit</td></tr><tr><td>HSC70</td><td>Santa Cruz</td><td>sc-7298</td><td>mouse</td></tr><tr><td>Lamin&amp;beta;</td><td>Santa Cruz</td><td>Sc-6261</td><td>goat</td></tr><tr><td>Prohibitin</td><td>Santa Cruz</td><td>sc-28259</td><td>rabbit</td></tr><tr><td>PSD95</td><td>Millipore</td><td>MAB1596</td><td>mouse</td></tr><tr><td>SNAP25</td><td>AbCam</td><td>ab5666-100</td><td>rabbit</td></tr><tr><td>Synaptophysin</td><td>Millipore</td><td>MAB368</td><td>mouse</td></tr><tr><td>&amp;beta;-3-Tubulin</td><td>Santa Cruz</td><td>sc-80016</td><td>mouse</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td colspan="4">&lt;p class=&quot;jove_content&quot;&gt; &lt;/p&gt;</td></tr></tbody></table>

preparation of synaptoneurosomes from mouse cortex using a discontinuous percoll-sucrose density gradient

Preparation of Synaptoneurosomes from Mouse Cortex using a Discontinuous Percoll-Sucrose Density Gradient

Synaptoneurosomes (SNs) are obtained after homogenization and fractionation of mouse brain cortex. They are resealed vesicles or isolated terminals that break away from axon terminals when the cortical tissue is homogenized. The SNs retain pre- and postsynaptic characteristics, which makes them useful in the study of synaptic transmission. They retain the molecular machinery used in neuronal signaling and are capable of uptake, storage, and release of neurotransmitters.
The production and isolation of active SNs can be problematic using medias like Ficoll, which can be cytotoxic and require extended centrifugation due to high density, and filtration and centrifugation methods, which can result in low activity due to mechanical damage of the SNs. However, the use of discontinuous Percoll-sucrose density gradients to isolate SNs provides a rapid method to produce good yields of translationally active SNs. The Percoll-sucrose gradient method is quick and gentle as it employs isotonic conditions, has fewer and shorter centrifugation spins and avoids centrifugation steps that pellet SNs and cause mechanical damage.

A method to prepare translationally active, intact synaptoneurosomes (SNs) from mouse brain cortex is described. The method uses a discontinuous Percoll-sucrose density gradient allowing for the quick preparation of active SNs.

Synaptoneurosomes (SNs) are obtained after homogenization and fractionation of mouse brain cortex. They are resealed vesicles or isolated terminals that ...

The overall goal of this procedure is to prepare translationally active synap to neuro zones from mouse brain cortex, using a discontinuous peral sucrose density gradient. This is accomplished by first collecting and homogenizing the mouse cortices, and then centrifuging the homogenate. Following this, apply the centrifuge homogenate, or sup natant to the preport per co sucrose gradients.The final step is to centrifuge and collect the synaptosome fraction. Ultimately, western blot analysis and S 35 methionine incorporation experiments show that the synaptosome fraction is synaptically enriched and translationally active. The main advantages of this technique over existing methods like centrifugation and filtration, our first mechanical damage is avoided by utilizing density gradients, and second per call has low toxicity towards cells in their constituents.Generally, individuals new to this method will struggle with this protocol because timing plays a key role. Once the cortis have been harvested, the procedure should be rapidly completed. To begin the procedure, prepare the 3, 10, 15, and 23%gradient layers by adding the respective amounts of SIP as described in the accompanying manuscript to GM buffer and mix well.Pour gradient layers by pipetting two milliliters from each of the 23 15, 10, and 3%isosmotic perol solutions into the Beckman centrifuge tubes with caps. Using a P 1000 pipette, the interface between the layers should be clearly discernible with no mixing of the layers. Store the gradients of four degrees Celsius for at least 20 minutes before use.Before starting this procedure, prepare other necessary solutions as described in the accompanying manuscript. Euthanize the mouse at the age of P 13 to P 21 and prepare it for dissection by spraying the back of the neck and head with 70%ethanol. Then use a pair of sharp scissors to cut through the spinal cord at the base of the skull.Next, remove the skin from the top of the skull. Cut the skull laterally between the parietal bone and the inter parietal bone, or between the cerebrum and the cortex regions. After that, cut the skull from the base to the nose along the sagittal suture.At this step, carefully remove the soft parietal bone by pulling each hemisphere to the side. After that, insert the spatula into the brain above the cerebellum to scoop out the cortex. Place it in ice cold GM buffer, and repeat the procedures again for collecting more cortices.Now rinse the cortices in ice cold GM buffer. Then transfer two cortices to a glass down homogenizer containing five milliliters of cold GM buffer. Gently homogenize the cortices with five to 10 strokes of pestle A, the loose pestle followed by five to 10 strokes of pestle B, the type pestle.The number of strokes varies depending on the individual homogenizer. Transfer the homogenate to a 15 milliliter conical tube. Centrifuge it at 1000 times.Gravity for 10 minutes at four degrees Celsius in an Allegra six KR centrifuge to pellet cellular debris and nuclei. Layer two milliliters of the supinate for each per or sucrose gradient with one gradient for each whole cortex and cap the tubes. Then centrifuge them in a fixed angle rotor at 32, 500 times gravity for five minutes at four degrees Celsius in a Beckman J 2 21 centrifuge.Using appropriate adapters when completed, the gradient should give a strong SN band pipette off and discard the solution above the SN band. Using a glass pasta pipette pipettes off the SN band at the 15 to 23%interface, one gradient generally gives about 0.9 to 1.1 milliliters of sns. After that, transfer it to a conical tube and store on ice.Then adjust the salt concentration of the SNS by adding one 10th volume of 10 times stimulation buffer. Optionally add 1000 times calcium chloride to give a final concentration of 12 ano molar. Next, add one millimolar TTX stock to give a final concentration of one micromolar to suppress non-specific excitation.The next step is to use the SNS in the applicable downstream application, such as protein translation studies. For other applications, SN lysate may be cleaned up or concentrated using the Pierce SDS page sample prep kit. The protein concentration of the SNS can be determined using the micro BCA protein assay kit.Here is an example of six bands or fractions. When the mouse cortex was homogenized and separated on discontinuous perol sucrose gradients, the enriched SNS were contained in Band five at the 23 to 15%interface, this band was removed and examined by electron microscopy. An example of intact synaptic vesicles and the preservation of pre-synaptic and post-synaptic elements are shown here in the western blot.An increase in synaptic markers and a decrease in impurities in the SN band from a discontinuous per sucrose gradient are shown to confirm that the increase in S 35 methionine incorporation upon addition of glutamate is due to de novo protein synthesis. 40 micromolar anesa mycin a protein synthesis inhibitor was added. This figure shows the marked decrease in S 35 methionine incorporation in the presence of anso mycin with or without glutamate present when compared to the basal levels.Thus, the discontinuous peral sucrose gradients quickly produce highly active and relatively pure SNS that can be used to study protein translation. This figure shows that band five from the discontinuous per core sucrose gradient contain the highest levels of de novo protein synthesis. The SNS prepared by passing homogenized cortex through a series of filters with decreasing pore size and then centrifuging over a discontinuous per core sucrose gradient for comparison contain more broken membranes and less whole SNS than SNS prepared from the discontinuous perol sucrose gradient method SNS prepared using the discontinuous peral sucrose gradient method are shown to contain more de novo protein synthesis activity then SNS prepared using the filtration method.SNS prepared from younger mice are shown to have greater translational activity than older mice. After watching this video, you should have a good idea of how to isolate translationally active synaptic neuro zones by homogenizing mouse cortices centrifuging the homogenate in a swinging bucket rotor, centrifuging the supernatant over a per call sucrose gradient in a fixed angle rotor, and then collecting the resulting synaptic neuro zone band.

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Neuroscience

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Preparation of Dissociated Mouse Cortical Neuron Cultures

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Source: Lunelli, L., et. al. Peering at Brain Polysomes with Atomic Force Microscopy. J. Vis. Exp.(2016).This video demonstrates the imaging of polysomes using atomic force microscopy. A nickel-ions-coated mica sheet anchors RNA with attached ribosomes. The sample is washed, dried, and mounted onto the microscope stage. The cantilever tip scans the sample surface, recording laser deflections to map the topology. Software is used to correct distortions and visualize high-resolution polysome...

 Imaging Polysomes Isolated from a Mouse Brain Using Atomic Force Microscopy

This article details the enrichment of proteins associated with the synaptic plasma membrane by ultracentrifugation on a discontinuous sucrose gradient. The subsequent preparation of post-synaptic density proteins is also described. Protein preparations are suitable for western blotting or 2D DIGE analysis.

Preparation of Synaptic Plasma Membrane and Postsynaptic Density Proteins Using a Discontinuous Sucrose Gradient

While ribosome structure has been extensively characterized, the organization of polysomes is still understudied. To overcome this lack of knowledge, we present here a detailed preparation protocol for accurate imaging of mammalian polysomes by atomic force microscopy (AFM) in air and liquid.

Peering at Brain Polysomes with Atomic Force Microscopy

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High Resolution Quantitative Synaptic Proteome Profiling of Mouse Brain Regions After Auditory Discrimination Learning

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

A protocol for the isolation of primary microglia from murine brains is presented. This technique aids in furthering the current understanding of neurological conditions. Density gradient centrifugation and magnetic separation are combined to produce sufficient yield of a highly pure sample. Furthermore, we outline the steps for characterization of microglia.

Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation

Challenging young neurons in new brain regions can reveal important insights into how the environment sculpts neuronal fate and maturation. This protocol describes a procedure to harvest interneuron precursors from specific brain regions and transplant them either homotopically or heterotopically into the brain of postnatal pups.

Homochronic Transplantation of Interneuron Precursors into Early Postnatal Mouse Brains

The development of the mammalian brain requires proper control of gene expression at the level of translation. Here, we describe a polysome profiling system with an easy-to-assemble sucrose gradient-making and fractionation platform to assess the translational status of mRNAs in the developing brain.

Analysis of Translation in the Developing Mouse Brain using Polysome Profiling

This protocol presents a robust, detailed method to obtain highly pure synaptosomes, synaptic vesicles, and other synaptic fractions from the mouse brain. This method enables the evaluation of synaptic processes, including the biochemical analysis of protein localization and function with compartmental resolution.

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

Learn to prepare translationally active synaptoneurosomes from mouse cortex using a discontinuous Percoll-sucrose density gradient. This method minimizes mechanical damage and enables reliable study of synaptic protein synthesis.

Preparation of Synaptoneurosomes from Mouse Cortex using a Discontinuous Percoll-Sucrose Density Gradient

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