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; varying GM buffer, SIP volumes for chromatography analysis." src="/files/ftp_upload/3196/3196table0.jpg" data-altupdatedat="1747911167000" 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: bands show membranes, myelin, and organelles like nuclei, Golgi." src="/files/ftp_upload/3196/3196table1.jpg" data-altupdatedat="1747911167000" data-alt="Table 1"> 
Table 1. Constituents of the bands from the discontinuous Percoll-gradient fractionations of homogenized mouse cortex.3,5
<img alt="Western blot analysis; protein band intensity; structural, synaptic, and mitochondrial markers." src="/files/ftp_upload/3196/3196table2.jpg" data-altupdatedat="1747911167000" 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, homogenization, centrifugation in Percoll gradient, diagram of SN band extraction." src="/files/ftp_upload/3196/3196scheme1.jpg" data-altupdatedat="1747911167000" 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 tube diagram with 3%-23% layers, protein band separation analysis." src="/files/ftp_upload/3196/3196fig1.jpg" data-altupdatedat="1747911167000" 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, cellular structures; detailed microscopic image for ultrastructural analysis." src="/files/ftp_upload/3196/3196fig2.jpg" data-altupdatedat="1747911167000" 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 results; protein expression analysis; β-Actin, GFAP, Synaptophysin; bands 1-6." src="/files/ftp_upload/3196/3196fig3.jpg" data-altupdatedat="1747911167000" 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="Protein electrophoresis and relative S35-Met incorporation bar chart; drug effects analyzed." src="/files/ftp_upload/3196/3196fig4.jpg" data-altupdatedat="1747911167000" 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 gel and bar chart showing S35-Met incorporation analysis for Glu treatment." src="/files/ftp_upload/3196/3196fig5.jpg" data-altupdatedat="1747911167000" 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; test tubes, chromatography result, visible separation bands." src="/files/ftp_upload/3196/3196fig6.jpg" data-altupdatedat="1747911167000" 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="Electrophoresis and bar graph showing S35-Met incorporation; protein analysis and filtration results." src="/files/ftp_upload/3196/3196fig7.jpg" data-altupdatedat="1747911167000" 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="Gel electrophoresis and bar graph showing S35-Met incorporation in protein synthesis analysis." src="/files/ftp_upload/3196/3196fig8.jpg" data-altupdatedat="1747911167000" 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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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enrichment+of+Presynaptic+and+Postsynaptic+Markers+by+Size-Based+Gating+Analysis+of+Synaptosome+Preparations+From+Rat+and+Human+Cortex.">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>Haj&#243;s, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+method+for+the+preparation+of+synaptosomal+fractions+in+high+purity.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptosome+Proteomics.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+that+protein+constituents+of+postsynaptic+membrane+specializations+are+locally+synthesized:+analysis+of+proteins+synthesized+within+synaptosomes.">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., Catsimpoolas, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isopycnic+separation+of+cells+and+cell+organelles+by+centrifugation+and+modified+colloidal+silica+gradients.">Isopycnic separation of cells and cell organelles by centrifugation and modified colloidal silica gradients.</a> Methods of Cell Separation. , 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+affinity+uptake+of+L-glutamate+and+&amp;gamma;-aminobutyric+acid+in+Drosophila+melanogaster.">High affinity uptake of L-glutamate and &amp;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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fractionation+of+granulocytes+from+whole+human+blood+by+centrifugation.+Practical+hints.">Fractionation of granulocytes from whole human blood by centrifugation. Practical hints.</a> Romanian J. Biophys. 14, 53-58 (2004).</li><li>Vlasselaer, P., Van Palathumpat, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+separation+composition,+kit+and+method.">Cell separation composition, kit and method.</a> US patent. , (1997).</li></ol>

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

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.

Watch this Scientific Journal Video about Preparation of Synaptoneurosomes from Mouse Cortex using a Discontinuous Percoll-Sucrose Density Gradient at JoVE.com

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

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 ...

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Research

JoVE Journal

Neuroscience

31.5K Views.  University of Wisconsin. Een methode om translationeel actief, intact synaptoneurosomes (SNS) voor te bereiden van de hersenen van muizen cortex wordt beschreven. De methode maakt gebruik van een discontinue Percoll-sucrose dichtheidsgradiënt waardoor voor de snelle bereiding van actieve SNS.

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

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

Assessing the synaptic protein composition and function constitutes an important challenge in neuroscience. However, it is not easy to evaluate neurotransmission that occurs within synapses because it is highly regulated by dynamic protein-protein interactions and phosphorylation events. Accordingly, when any method is used to study synaptic transmission, a major goal is to preserve these transient physiological modifications. Here, we present a brain membrane fractionation protocol that represents a robust procedure to isolate proteins belonging to different synaptic compartments. In other words, the protocol describes a biochemical methodology to carry out protein enrichment from presynaptic, postsynaptic, and extrasynaptic compartments. First, synaptosomes, or synaptic terminals, are obtained from neurons that contain all synaptic compartments by means of a discontinuous sucrose gradient. Of note, the quality of this initial synaptic membrane preparation is critical. Subsequently, the isolation of the different subsynaptic compartments is achieved with light solubilization using mild detergents at differential pH conditions. This allows for separation by gradient and isopycnic centrifugations. Finally, protein enrichment at the different subsynaptic compartments (i.e., pre-, post- and extrasynaptic membrane fractions) is validated by means of immunoblot analysis using well-characterized synaptic protein markers (i.e., SNAP-25, PSD-95, and synaptophysin, respectively), thus enabling a direct assessment of the synaptic distribution of any particular neuronal protein.

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

Here, we present a brain membrane fractionation protocol that represents a robust procedure to isolate proteins belonging to different synaptic compartments.

Assessing the synaptic protein composition and function constitutes an important challenge in neuroscience. However, it is not easy to evaluate ...

Biochemistry

Preparation of Dissociated Mouse Cortical Neuron Cultures

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures can be used for a variety of applications including immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging, protein and/or RNA isolation. These cultures also provide a platform to study the neuronal development of transgenic animals that carry a late embryonic or postnatal lethal gene mutation. The procedure is relatively straight forward, requires some experience in tissue culture technique and should not take longer than two to three hours if you are properly prepared. Careful separation of the cortical rind from the thalamo-cortical fiber tract will reduce the number of unwanted non-neuronal cells. To increase yields of neuronal cells triturate the pieces of the cortical tissue gently after the enzyme incubation step. This is imperative as it prevents unnecessary injury to cells and premature neuronal cell death. Since these cultures are maintained in the absence of glia feeder cells, they also offer an added advantage of growing cultures enriched in neurons.

This video shows a procedure for generating neuronal cultures from late embryo and early postnatal mouse cortex. These cultures can be used for immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging and provide a platform to study the neuronal development of transgenic animals that carry a postnatal lethal gene mutation.

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures ...

Biology

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

Neuronal subcellular fractionation techniques allow the quantification of proteins that are trafficked to and from the synapse. As originally described in the late 1960&#8217;s, proteins associated with the synaptic plasma membrane can be isolated by ultracentrifugation on a sucrose density gradient. Once synaptic membranes are isolated, the macromolecular complex known as the post-synaptic density can be subsequently isolated due to its detergent insolubility. The techniques used to isolate synaptic plasma membranes and post-synaptic density proteins remain essentially the same after 40 years, and are widely used in current neuroscience research. This article details the fractionation of proteins associated with the synaptic plasma membrane and post-synaptic density using a discontinuous sucrose gradient. Resulting protein preparations are suitable for western blotting or 2D DIGE analysis.

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.

Neuronal subcellular fractionation techniques allow the quantification of proteins that are trafficked to and from the synapse. As originally described in ...

Peering at Brain Polysomes with Atomic Force Microscopy

The translational machinery, i.e., the polysome or polyribosome, is one of the biggest and most complex cytoplasmic machineries in cells. Polysomes, formed by ribosomes, mRNAs, several proteins and non-coding RNAs, represent integrated platforms where translational controls take place. However, while the ribosome has been widely studied, the organization of polysomes is still lacking comprehensive understanding. Thus much effort is required in order to elucidate polysome organization and any novel mechanism of translational control that may be embedded. Atomic force microscopy (AFM) is a type of scanning probe microscopy that allows the acquisition of 3D images at nanoscale resolution. Compared to electron microscopy (EM) techniques, one of the main advantages of AFM is that it can acquire thousands of images both in air and in solution, enabling the sample to be maintained under near physiological conditions without any need for staining and fixing procedures. Here, a detailed protocol for the accurate purification of polysomes from mouse brain and their deposition on mica substrates is described. This protocol enables polysome imaging in air and liquid with AFM and their reconstruction as three-dimensional objects. Complementary to cryo-electron microscopy (cryo-EM), the proposed method can be conveniently used for systematically analyzing polysomes and studying their organization.

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.

The translational machinery, i.e., the polysome or polyribosome, is one of the biggest and most complex cytoplasmic machineries in cells. Polysomes, ...

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

The molecular synaptic mechanisms underlying auditory learning and memory remain largely unknown. Here, the workflow of a proteomic study on auditory discrimination learning in mice is described. In this learning paradigm, mice are trained in a shuttle box Go/NoGo-task to discriminate between rising and falling frequency-modulated tones in order to avoid a mild electric foot-shock. The protocol involves the enrichment of synaptosomes from four brain areas, namely the auditory cortex, frontal cortex, hippocampus, and striatum, at different stages of training. Synaptic protein expression patterns obtained from trained mice are compared to na&#239;ve controls using a proteomic approach. To achieve sufficient analytical depth, samples are fractionated in three different ways prior to mass spectrometry, namely 1D SDS-PAGE/in-gel digestion, in-solution digestion and phospho-peptide enrichment. 
High-resolution proteomic analysis on a mass spectrometer and label-free quantification are used to examine synaptic protein profiles in phospho-peptide-depleted and phospho-peptide-enriched fractions of synaptosomal protein samples. A commercial software package is utilized to reveal proteins and phospho-peptides with significantly regulated relative synaptic abundance levels (trained/na&#239;ve controls). Common and differential regulation modes for the synaptic proteome in the investigated brain regions of mice after training were observed. Subsequently, meta-analyses utilizing several databases are employed to identify underlying cellular functions and biological pathways.

The identification of molecules and pathways controlling synaptic plasticity and memory is still a major challenge in neuroscience. Here, a workflow is described addressing the relative quantification of synaptic proteins supposedly involved in the molecular reorganization of synapses during learning and memory consolidation in an auditory learning paradigm.

The molecular synaptic mechanisms underlying auditory learning and memory remain largely unknown. Here, the workflow of a proteomic study on auditory ...

Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation

Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has revealed microglia to be dynamic, capable of assuming both pro-inflammatory and anti-inflammatory phenotypes. Both M1 (pro-inflammatory) and M2 (pro-reparative) phenotypes play an important role in neuroinflammatory conditions such as perinatal brain injury, and exhibit differing functions in response to certain environmental stimuli. The modulation of microglial activation has been noted to confer neuroprotection thus suggesting microglia may have therapeutic potential in brain injury. However, more research is required to better understand the role of microglia in disease, and this protocol facilitates that. The protocol described below combines a density gradient centrifugation process to reduce cellular debris, with magnetic separation, producing a highly pure sample of primary microglial cells that can be used for in vitro experimentation, without the need for 2-3 weeks culturing. Additionally, the characterization steps yield robust functional data about microglia, aiding studies to better our understanding of the polarization and priming of these cells, which has strong implications in the field of regenerative medicine.

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.

Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has ...

Analysis of Translation in the Developing Mouse Brain using Polysome Profiling

The proper development of the mammalian brain relies on a fine balance of neural stem cell proliferation and differentiation into different neural cell types. This balance is tightly controlled by gene expression that is fine-tuned at multiple levels, including transcription, post-transcription and translation. In this regard, a growing body of evidence highlights a critical role of translational regulation in coordinating neural stem cell fate decisions. Polysome fractionation is a powerful tool for the assessment of mRNA translational status at both global and individual gene levels. Here, we present an in-house polysome profiling pipeline to assess translational efficiency in cells from the developing mouse cerebral cortex. We describe the protocols for sucrose gradient preparation, tissue lysis, ultracentrifugation and fractionation-based analysis of mRNA translational status.

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.

The proper development of the mammalian brain relies on a fine balance of neural stem cell proliferation and differentiation into different neural cell ...

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

Synaptic terminals are the primary sites of neuronal communication. Synaptic dysfunction is a hallmark of many neuropsychiatric and neurological disorders. The characterization of synaptic sub-compartments by biochemical isolation is, therefore, a powerful method to elucidate the molecular bases of synaptic processes, both in health and disease. This protocol describes the isolation of synaptic terminals and synaptic sub-compartments from mouse brains by subcellular fractionation. First, sealed synaptic terminal structures, known as synaptosomes, are isolated following brain tissue homogenization. Synaptosomes are neuronal pre- and post-synaptic compartments with pinched-off and sealed membranes. These structures retain a metabolically active state and are valuable for studying synaptic structure and function. The synaptosomes are then subjected to hypotonic lysis and ultracentrifugation to obtain synaptic sub-compartments enriched for synaptic vesicles, synaptic cytosol, and synaptic plasma membrane. Fraction purity is confirmed by electron microscopy and biochemical enrichment analysis for proteins specific to sub-synaptic compartments. The presented method is a straightforward and valuable tool for studying the structural and functional characteristics of the synapse and the molecular etiology of various brain disorders.

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.

Synaptic terminals are the primary sites of neuronal communication. Synaptic dysfunction is a hallmark of many neuropsychiatric and neurological ...

Mononuclear Cell Isolation from Mouse Brain: A Density Gradient Centrifugation Technique to Isolate Brain Resident Mononuclear Cells

Source: Ji, Z. et al. <a href="https://www.jove.com/v/61050/flow-cytometric-analysis-lymphocyte-infiltration-central-nervous">Flow Cytometric Analysis of Lymphocyte Infiltration in Central Nervous System during Experimental Autoimmune Encephalomyelitis.</a> J. Vis. Exp. (2020)
In this video, we perform the isolation of mononuclear cells from a mouse&#8217;s brain using density gradient centrifugation. The isolated mononuclear cell suspension can be used for further downstream analysis.

Source: Ji, Z. et al. Flow Cytometric Analysis of Lymphocyte Infiltration in Central Nervous System during Experimental Autoimmune Encephalomyelitis. J. ...

JoVE Encyclopedia of Experiments

Cancer Research

Cancers of the Nervous System

Ex vivo studies

 Imaging Polysomes Isolated from a Mouse Brain Using Atomic Force Microscopy

Source: Lunelli, L.,&#160;et. al. Peering at Brain Polysomes with Atomic Force Microscopy.&#160;J. Vis. Exp.(2016).This video demonstrates the imaging of ...

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

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