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&deg;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&deg;C.</li>
 <li>Pre-cool centrifuges to 4&deg;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="Table 0" src="/files/ftp_upload/3196/3196table0.jpg" />
<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&deg;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&deg;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&deg;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 &mu;M 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 &mu;L, 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 &mu;M glutamate/10 &mu;M 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 &mu;M 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="Table 1" src="/files/ftp_upload/3196/3196table1.jpg" /> 
Table 1. Constituents of the bands from the discontinuous Percoll-gradient fractionations of homogenized mouse cortex.3,5
<img alt="Table 2" src="/files/ftp_upload/3196/3196table2.jpg" /> 
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&ge;0.2 (+), 0.2&ge;0.8 (++), or &gt;0.8 (+++) fold greater than protein present in the supernatant (S, centrifuged cortex homogenate), representative of n=3.
<img alt="Scheme 1" src="/files/ftp_upload/3196/3196scheme1.jpg" /> 
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="Figure 1" src="/files/ftp_upload/3196/3196fig1.jpg" /> 
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="Figure 2" src="/files/ftp_upload/3196/3196fig2.jpg" /> 
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 &mu;m.
<img alt="Figure 3" src="/files/ftp_upload/3196/3196fig3.jpg" /> 
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 &beta;-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 &beta;3-Tubulin (neuron-specific). The bands were visualized using a Storm 860 Phosphorimager, representative of n=3.
 
<img alt="Figure 4" src="/files/ftp_upload/3196/3196fig4.jpg" /> 
Figure 4. SNs are active and exhibit de novo protein synthesis via [35S]-Met incorporation. SNs were pre-equilibrated to 37&deg;C for 10 min. Then the SNs were untreated or pretreated for 15 min with 40 &mu;M anisomycin or 1 &mu;M juglone at 37&deg;C followed by addition of [35S]-Met, 20 &mu;l Express Pro Label Mix S35 Easy Tag, plus or minus Glu at 37&deg;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, &plusmn;SEM.
<img alt="Figure 5" src="/files/ftp_upload/3196/3196fig5.jpg" /> 
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&deg;C for 10 min. Then, [35S]-Met, Express Pro Label Mix S35 Easy Tag, was added, plus or minus Glu at 37&deg;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, &plusmn;SEM.
<img alt="Figure 6" src="/files/ftp_upload/3196/3196fig6.jpg" /> 
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="Figure 7" src="/files/ftp_upload/3196/3196fig7.jpg" /> 
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&deg;C for 10 min. Then, [35S]-Met, Express Pro Label Mix S35 Easy Tag, was added, plus or minus Glu at 37&deg;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, &plusmn;SEM.
<img alt="Figure 8" src="/files/ftp_upload/3196/3196fig8.jpg" /> 
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&deg;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, &plusmn;SEM. SNs from P14 mice were at least 3-fold more active than P85 mice.

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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 border="1">
	<tbody>
		<tr>
			<th>
				Name of the reagent</th>
			<th>
				Company</th>
			<th>
				Catalogue number</th>
			<th>
				Comments (optional)</th>
		</tr>
		<tr>
			<td>Micro BCA Protein Assay Kit</td>
			<td>Pierce</td>
			<td>23235</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Fisher</td>
			<td>C79-500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CO2 gas</td>
			<td>Airgas (UW-MDS)</td>
			<td>CD 50</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>RPI</td>
			<td>E57020</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>EtOH</td>
			<td>Fisher</td>
			<td>A407SK-4</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Fisher</td>
			<td>A142-212</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Percoll</td>
			<td>GE Healthcare</td>
			<td>17-0891-01</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Fisher</td>
			<td>P285-500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PierceSDS-PAGE Sample Prep Kit</td>
			<td>Pierce</td>
			<td>89888</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>RPI</td>
			<td>S23020</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Fisher</td>
			<td>BP328-500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Na2PHO4</td>
			<td>Fisher</td>
			<td>S381-500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>RPI</td>
			<td>S24060</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>RPI</td>
			<td>T60040</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tetrodotoxin</td>
			<td>Sigma</td>
			<td>T5651</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Express Pro Label Mix S35 Easy Tag</td>
			<td>Perkin Elmer</td>
			<td>NEG772</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<th>
				Equipment</th>
			<th>
				Company</th>
			<th>
				Catalogue number</th>
			<th>
				Comments (optional)</th>
		</tr>
		<tr>
			<td>Dissection tools</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dounce homogenizer, 7 mL (comes with two glass pestles labled " A" and "B")</td>
			<td>Wheaton</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>P1000 Gilson Pipetman</td>
			<td>Gilson</td>
			<td>F123602</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Allegra 6KR Centrifuge</td>
			<td>Beckman Coulter</td>
			<td>366830</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>GH 3.8 Rotor, Swinging bucket rotor</td>
			<td>Beckman Coulter</td>
			<td>360581</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Beckman J2-21 Centrifuge</td>
			<td>Beckman</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Beckman tubes with caps</td>
			<td>Beckman</td>
			<td>355672</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>White walled adapters</td>
			<td>Beckman</td>
			<td>342327</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Blue walled adapters</td>
			<td>Beckman</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>JA-17 Rotor, Fixed-angle rotor</td>
			<td>Beckman</td>
			<td>369691</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>
Table of antibodies used for western blots:
<table border="1">
	<tbody>
		<tr>
			<th>
				Name of Antibody</th>
			<th>
				Company</th>
			<th>
				Catalogue number</th>
			<th>
				Host Species</th>
			<th>
				Dilution Factor</th>
		</tr>
		<tr>
			<td>&beta;-Actin</td>
			<td>Sigma</td>
			<td>A5441</td>
			<td>mouse</td>
			<td>1:2000</td>
		</tr>
		<tr>
			<td>GFAP</td>
			<td>Santa Cruz</td>
			<td>sc-65343</td>
			<td>mouse</td>
			<td>1:200</td>
		</tr>
		<tr>
			<td>GP73</td>
			<td>Santa Cruz</td>
			<td>Sc-134509</td>
			<td>rabbit</td>
			<td>1:200</td>
		</tr>
		<tr>
			<td>HSC70</td>
			<td>Santa Cruz</td>
			<td>sc-7298</td>
			<td>mouse</td>
			<td>1:200</td>
		</tr>
		<tr>
			<td>Lamin&beta;</td>
			<td>Santa Cruz</td>
			<td>Sc-6261</td>
			<td>goat</td>
			<td>1:200</td>
		</tr>
		<tr>
			<td>Prohibitin</td>
			<td>Santa Cruz</td>
			<td>sc-28259</td>
			<td>rabbit</td>
			<td>1:200</td>
		</tr>
		<tr>
			<td>PSD95</td>
			<td>Millipore</td>
			<td>MAB1596</td>
			<td>mouse</td>
			<td>5 &mu;g/&mu;L</td>
		</tr>
		<tr>
			<td>SNAP25</td>
			<td>AbCam</td>
			<td>ab5666-100</td>
			<td>rabbit</td>
			<td>1:2000</td>
		</tr>
		<tr>
			<td>Synaptophysin</td>
			<td>Millipore</td>
			<td>MAB368</td>
			<td>mouse</td>
			<td>1:500</td>
		</tr>
		<tr>
			<td>&beta;-3-Tubulin</td>
			<td>Santa Cruz</td>
			<td>sc-80016</td>
			<td>mouse</td>
			<td>1:200</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 ...

preparation-synaptoneurosomes-from-mouse-cortex-using-discontinuous

Research

JoVE Journal

Neuroscience

31.4K Aufrufe.  University of Wisconsin. Eine Methode, um translationsaktiven, intakte Synaptoneurosomen (SNS) von der Maus Hirnrinde vorbereiten beschrieben. Das Verfahren nutzt eine diskontinuierliche Percoll-Saccharose-Dichtegradienten Dies ermöglicht die schnelle Erstellung von aktiven SNS.

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

Chronic Imaging of Mouse Visual Cortex Using a Thinned-skull Preparation

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The technique presented here allows the imaging of the same area of the brain at several time points (chronic imaging) with microscopic resolution allowing the tracking of dendritic spines which are the small structures that represent the majority of postsynaptic excitatory sites in the CNS. The ability to clearly resolve fine cortical structures over several time points has many advantages, specifically in the study of brain plasticity in which morphological changes at synapses and circuit remodeling may help explain underlying mechanisms. In this video and supplementary material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation. The thinned-skull preparation is a minimally invasive approach, which avoids potential damage to the dura and/or cortex, thus reducing the onset of an inflammatory response. When this protocol is performed correctly, it is possible to clearly monitor changes in dendritic spine characteristics in the intact brain over a prolonged period of time.

In this video and supplemental material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation.

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The ...

Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of intracellular events that lead to closure of cyclic-nucleotide-gated channels in the cell membrane. The resulting change in membrane potential leads in turn to reductions in the amount of neurotransmitter release from both rod and cone synaptic terminals. To measure how the light-evoked change in photoreceptor membrane potential leads to downstream activity in the retina, scientists have made electrophysiological recordings from retinal slice preparations for decades1,2. In the past these slices have been cut manually with a razor blade on retinal tissue that is attached to filter paper; in recent years another method of slicing has been developed whereby retinal tissue is embedded in low gelling temperature agar and sliced in cool solution with a vibrating microtome3,4. This preparation produces retinal slices with less surface damage and very robust light-evoked responses. Here we document how this procedure can be done under infrared light to avoid bleaching the visual pigment.

Here we describe a procedure for generating dark-adapted slices of the mouse retina for electrophysiological recordings.

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of ...

Isolating LacZ-expressing Cells from Mouse Inner Ear Tissues using Flow Cytometry

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues presents a unique challenge because of the paucity of cells of interest and few transgenic reporter mouse models. Here, we describe a protocol using fluorescence-conjugated probes to selectively label LacZ-positive cells from the neonatal cochleae.
The most common underlying pathology of sensorineural hearing loss is the irreversible damage and loss of cochlear sensory hair cells, which are required to transduce sound waves to neural impulses. Recent evidence suggests that the murine auditory and vestibular organs harbor stem/progenitor cells that may have regenerative potential1,2. These findings warrant further investigation, including identifying specific cell types with stem/progenitor cell characteristics. The Wnt signaling pathway has been demonstrated to play a critical role in maintaining stem/progenitor cell populations in several organ systems3-7. We have recently identified Wnt-responsive Axin2-expressing cells in the neonatal cochlea, but their function is largely unknown8.
To better understand the behavior of these Wnt-responsive cells in vitro, we have developed a method of isolating Axin2-expressing cells from cochleae of Axin2-LacZ reporter mice9. Using flow cytometry to isolate Axin2-LacZ positive cells from the neonatal cochleae, we could in turn execute a variety of experiments on live cells to interrogate their behavior as stem/progenitor cells. Here, we describe in detail the steps for the microdissection of neonatal cochlea, dissociation of these tissues, labeling of the LacZ-positive cells using a fluorogenic substrate, and cell sorting. Techniques for dissociating cochleae into single cells and isolating cochlear cells via flow cytometry have been described2,10-12. We have made modifications to these techniques to establish a novel protocol to isolate LacZ-expressing cells from the neonatal cochlea.

Flow cytometry is a powerful tool allowing for the isolation and study of specific cell populations. This protocol describes steps for isolating LacZ-expressing cells from cochlear tissues from neonatal transgenic mice. Dissociated cochlear cells were labeled using fluorescent-conjugated substrates of &#946;-galactosidase prior to separation via flow cytometry.

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as addition/removal of synapses, occur in an experience-dependent manner, providing the structural foundation of neuronal plasticity. As postsynaptic components of the most excitatory synapses in the cortex, dendritic spines are considered to be a good proxy of synapses. Taking advantages of mouse genetics and fluorescent labeling techniques, individual neurons and their synaptic structures can be labeled in the intact brain. Here we introduce a transcranial imaging protocol using two-photon laser scanning microscopy to follow fluorescently labeled postsynaptic dendritic spines over time in vivo. This protocol utilizes a thinned-skull preparation, which keeps the skull intact and avoids inflammatory effects caused by exposure of the meninges and the cortex. Therefore, images can be acquired immediately after surgery is performed. The experimental procedure can be performed repetitively over various time intervals ranging from hours to years. The application of this preparation can also be expanded to investigate different cortical regions and layers, as well as other cell types, under physiological and pathological conditions.

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Time-lapse imaging in the living animal provides valuable information on structural reorganization in the intact brain. Here, we introduce a thinned-skull preparation that allows transcranial imaging of fluorescently labeled synaptic structures in the living mouse cortex by two-photon microscopy.

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as ...

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 ...

Investigating the Function of Deep Cortical and Subcortical Structures Using Stereotactic Electroencephalography: Lessons from the Anterior Cingulate Cortex

Stereotactic Electroencephalography (SEEG) is a technique used to localize seizure foci in patients with medically intractable epilepsy. This procedure involves the chronic placement of multiple depth electrodes into regions of the brain typically inaccessible via subdural grid electrode placement. SEEG thus provides a unique opportunity to investigate brain function. In this paper we demonstrate how SEEG can be used to investigate the role of the dorsal anterior cingulate cortex (dACC) in cognitive control. We include a description of the SEEG procedure, demonstrating the surgical placement of the electrodes. We describe the components and process required to record local field potential (LFP) data from consenting subjects while they are engaged in a behavioral task. In the example provided, subjects play a cognitive interference task, and we demonstrate how signals are recorded and analyzed from electrodes in the dorsal anterior cingulate cortex, an area intimately involved in decision-making. We conclude with further suggestions of ways in which this method can be used for investigating human cognitive processes.

Stereotactic Electroencephalography (SEEG) is an operative technique used in epilepsy surgery to help localize seizure foci. It also affords a unique opportunity to investigate brain function. Here we describe how SEEG can be used to investigate cognitive processes in human subjects.

Stereotactic Electroencephalography (SEEG) is a technique used to localize seizure foci in patients with medically intractable epilepsy. This procedure ...

Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or postmitotic neurons. Later, some progenitors switch to a gliogenic fate, adding to the astrocyte and oligodendrocyte populations. Using time-lapse video-microscopy of primary cerebral cortex cell cultures, it is possible to study the cellular and molecular mechanisms controlling the mode of cell division and cell cycle parameters of progenitor cells. Similarly, the fate of postmitotic cells can be examined using cell-specific fluorescent reporter proteins or post-imaging immunocytochemistry. More importantly, all these features can be analyzed at the single-cell level, allowing the identification of progenitors committed to the generation of specific cell types. Manipulation of gene expression can also be performed using viral-mediated transfection, allowing the study of cell-autonomous and non-cell-autonomous phenomena. Finally, the use of fusion fluorescent proteins allows the study of symmetric and asymmetric distribution of selected proteins during division and the correlation with daughter cells fate. Here, we describe the time-lapse video-microscopy method to image primary cerebral cortex murine cells for up to several days and analyze the mode of cell division, cell cycle length and fate of newly generated cells. We also describe a simple method to transfect progenitor cells, which can be applied to manipulate genes of interest or simply label cells with reporter proteins.

Live imaging is a powerful tool to study cellular behaviors in real time. Here, we describe a protocol for time-lapse video-microscopy of primary cerebral cortex cells that allows a detailed examination of the phases enacted during the lineage progression from primary neural stem cells to differentiated neurons and glia.

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or ...

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 ...