Name: Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization (Video) | JoVE Journal
Uploaded: 2017-05-12T15:00:00.000+00:00
Duration: 9 min 49 s
Description: Learn brain membrane fractionation to isolate proteins from synaptic compartments in mouse hippocampus. This ex vivo method enables precise study of synaptic function in normal and pathological conditions.

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This work was supported by Ministerio de Econom&#236;a y Competitividad/Instituto de Salud Carlos III (SAF2014-55700-P, PCIN-2013-019-C03-03 and PIE14/00034), Instituci&#242; Catalana de Recerca i Estudis Avan&#231;ats (ICREA Academia-2010), and Agentschap voor Innovatie door Wetenschap en Technologie (SBO-140028) to F. C. Also, X. M, V. F.-D., and F. C. belong to the "Neuropharmacology and Pain" accredited research group (Generalitat de Catalunya,2014 SGR 1251). The work was also supported by the "Programa Pesquisador Visitante Especial-Ci&#234;ncia sem Fronteiras" from CAPES (Brazil) to F.C.

All animal experimental procedures were approved by the University of Barcelona Committee on Animal Use and Care (CEEA), in compliance with the guidelines described in the Guide for the Care and Use of Laboratory Animals11 and following the European Community, law 86/609/CCE, FELASA, and ARRIVE guidelines. Thus, mice are housed in standard cages, with ad libitum access to food and water, and are maintained under controlled standard conditions (12 h dark/light cycle starting at 7:30 AM, 22 &#176;C temperature, and 66% humidity).
1. Obtaining Mouse Brain Synaptosomes Using a Discontinuous Sucrose Gradient
Note: This method was reported previously10.
<ol>
	<li>Prepare fresh isolation buffer (IB), pH 7.4; 2 M sucrose; 1 M sucrose/0.1 mM CaCl2; and 0.1 mM CaCl2 (see Table 1). Chill the solutions on ice.</li>
	<li>Sacrifice five mice by cervical dislocation. Decapitate and rapidly remove the brain of each animal. Dissect the brain region of interest (i.e., the hippocampus, 0.25-0.35 g of fresh tissue) and place it in a 5 mL Potter-Elvehjem glass tube on ice with 1 mL of IB.</li>
	<li>Homogenize the tissue using a homogenizing stirrer (10 strokes at 700-900 rotations per min), preferentially in an ice bath to preclude sample warming.</li>
	<li>Place the homogenized tissue (1 mL) into a 15 mL tube containing 6 mL of 2 M sucrose and 2.5 mL of 0.1 mM CaCl2 at 4 &#176;C. Mix slowly by inverting the tube. 
	Note: The addition of calcium is essential to maintaining the adhesive interactions between organelles and thus to preserving the structure of the different &#34;densities.&#34;</li>
	<li>Place the solution in a 12 mL centrifuge tube and slowly add 2.5 mL of 1 M sucrose/0.1 mM CaCl2 on top of each tube to form the sucrose gradient. 
	Note: Label the position of the gradient interface on the centrifuge tube using a permanent marker.</li>
	<li>Weigh and equilibrate the centrifuge tubes with IB solution within their corresponding steel supports and with their closing lids.</li>
	<li>Centrifuge the tubes for 3 h at 100,000 x g and 4 &#176;C using a swinging bucket rotor centrifuge. Completely fill the rotor with all the steel supports (even empty, if required).</li>
	<li>Discard the top layer containing myelin. With a Pasteur pipette, collect the white ring between the 1.25-M and 1-M sucrose interphase corresponding to the synaptosome fraction.</li>
	<li>Dilute the synaptosomes with 9X their volume using IB in a centrifuge tube.</li>
	<li>Weigh and equilibrate the centrifuge tubes with IB solution within their corresponding steel supports and with their closing lids.</li>
	<li>Centrifuge the tubes for 30 min at 15,000 x g and 4 &#176;C using a swinging bucket rotor centrifuge.</li>
	<li>Discard the supernatant and resuspend the pellet with 1.1 mL of IB solution. Collect 100 &#181;L of this synaptosomal solution and centrifuge for 5 min at 11,000 x g and 4 &#186;C.</li>
	<li>Resuspend the synaptosomal pellet in 5% sodium dodecyl sulfate (SDS) and store this sample at -20 &#186;C. 
	Note: This sample will correspond to the total synaptosome fraction for further Western-blot analysis. The remaining synaptosomal fraction (~1 mL) can either be frozen down at -20 &#186;C until use or processed for subsequent subsynaptic fractionation. The synaptosomes or purified synapses represent between 1 and 2% of the total hippocampal volume12.</li>
</ol>
2. Pre-, Post- and Extrasynaptic-isolation
<ol>
	<li>Prepare 2x fresh solubilization buffer, pH 6.0; 1x solubilization buffer, pH 6.0; solubilization buffer, pH 8.0; and 0.1 mM CaCl2 (see Table 1). Chill the solutions on ice. 
	Note: The pH of these solutions should be accurately adjusted to achieve a good subsynaptic fractionation.</li>
	<li>Slowly dilute the 1-mL resuspended synaptosomal fraction (see step 1.12) with 5 mL of 0.1 mM CaCl2.</li>
	<li>Add the same volume (5 mL) of ice-cold 2x solubilization buffer, pH 6.0, and incubate for 50 min on ice under high agitation in a beaker on ice. 
	Note: While 1% of Triton X-100 at pH 6.0 solubilizes all plasma membrane proteins, it preserves proteins within the synaptic contacts or junctions (i.e., pre- and postsynaptic structures)4.</li>
	<li>Place the solution in a centrifuge tube. Weigh and equilibrate the tubes with equivalent volumes of 0.1 mM CaCl2 and 2x solubilization buffer solution, pH 6.0, within their corresponding steel supports and with their closing lids.</li>
	<li>Centrifuge the tubes for 30 min at 40,000 x g and 4 &#176;C using a swinging bucket rotor centrifuge. The resulting supernatant represents the extrasynaptic fraction, and the pellet corresponds to the synaptic junctions (i.e., pre- and postsynaptic fractions).</li>
	<li>Concentrate the supernatant containing the extrasynaptic fraction to a final 200 &#181;L volume using a 15 mL 10K filter tube centrifuged at 4,000 x g and 4 &#186;C.</li>
	<li>Precipitate the resulting concentrated extrasynaptic fraction (200 &#181;L) with 5 volumes (1 mL) of pre-chilled (-20 &#176;C) acetone overnight at -20 &#176;C.</li>
	<li>The next day, centrifuge the extrasynaptic fraction for 30 min at 18,000 x g and -15 &#176;C. After centrifugation, discard the acetone supernatant and dry the pellet to remove any trace of acetone.</li>
	<li>Finally, resuspend the pellet containing the extrasynaptic proteins with 200 &#181;L of 5% SDS. Sonicate the pellet, if required.</li>
	<li>Without disrupting it, carefully wash the pellet containing the pre- and postsynaptic fractions with 2 mL of 1x solubilization buffer, pH 6.0. Discard the buffer.</li>
	<li>Resuspend the pellet with 10 mL of 1x solubilization buffer, pH 8.0, using a glass Pasteur pipette. 
	Note: While 1% of Triton X-100 at pH 8.0 solubilizes the presynaptic specialization, it preserves the insoluble postsynaptic density4.</li>
	<li>Incubate the suspension for 50 min on ice under high agitation in a beaker on ice.</li>
	<li>Place the solution in a centrifuge tube. Weigh and equilibrate the tubes with 1x solubilization buffer solution, pH 8.0, within their corresponding steel supports and with their closing lids.</li>
	<li>Centrifuge the tubes for 30 min at 40,000 x g and 4 &#176;C using a swinging bucket rotor centrifuge; the resulting supernatant corresponds to the presynaptic fraction and the pellet to the postsynaptic fraction.</li>
	<li>Process the supernatant containing the presynaptic fraction, as described in steps 2.6-2.9.</li>
	<li>Resuspend the pellet containing postsynaptic fraction with 200 &#181;L of 5% SDS. Sonicate the pellet, if required.</li>
</ol>
3. Analyze the samples by immunoblot to validate the membrane fractionation
<ol>
	<li>Determine the amount of protein in each fraction (i.e., the total, extra-, pre- and post-synaptic fractions) using the bicinchoninic acid protein assay.</li>
	<li>Take 20 &#181;g of protein from each fraction and dilute it to a final volume of 50 &#181;L in SDS-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer. Boil for 5 min at 100 &#186;C.</li>
	<li>Separate the proteins by SDS-PAGE electrophoresis 10%, with a 4% concentrating gel under reducing conditions. Electrophorese at constant voltage of 80 V until the dye enters the lower gel and then increase the voltage to 120 V.</li>
	<li>Transfer the proteins to a PVDF membrane and block with IB blocking solution for 45 min at room temperature under continuous shaking.</li>
	<li>Incubate the membrane overnight at 4 &#186;C with the indicated primary antibody (i.e., anti-SNAP-25, anti-PSD-95, anti-synaptophysin, or anti-GPR37) diluted in IB blocking solution under continuous shaking.</li>
	<li>Wash the membrane three times (10 min each) with IB washing solution to eliminate unbound primary antibody.</li>
	<li>Incubate with the indicated secondary antibody for 90 min in dark conditions at room temperature under continuous shaking.</li>
	<li>Wash the membrane three times (10 min each) with IB washing solution to eliminate unbound secondary antibody.</li>
	<li>Incubate the membrane with chemiluminescent substrate (prepare the mix under dark conditions and following the 1:1 proportion of solution A and B provided by the manufacturer).</li>
	<li>Analyze the membrane in a chemiluminescent detection device.</li>
</ol>

<ol><li>Foster, M., Sherrington, C. S. <a target="_blank" href="https://archive.org/details/textbookofphysio1897fost">Part III: The Central Nervous System.</a> A Text Book of Physiology. , Macmillan: London. (1897).</li>
<li>Peters, A., Palay, S. L., Webster, H. D. F. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aPeters%2C+A+%22The+fine+structure+of+the+nervous+system%22">The fine structure of the nervous system</a>. , Oxford University Press. New York. (1991).</li>
<li>Harris, K. M., Weinberg, R. J. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/22357909">Ultrastructure of Synapses in the Mammalian Brain.</a> Cold Spring Harb. Perspect. Biol. 4, 005587-005587 (2012).</li>
<li>Phillips, G. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+presynaptic+particle+web%3A+ultrastructure%2C+composition%2C+dissolution%2C+and+reconstitution%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">The presynaptic particle web: ultrastructure, composition, dissolution, and reconstitution.</a> Neuron. 32, 63-77 (2001).</li>
<li>Pinheiro, P. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Solubilization+and+immunological+identification+of+presynaptic+alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic+acid+receptors+in+the+rat+hippocampus%5BTitle%5D)+AND+%22Neurosci.+Lett%22%5BJournal%5D)">Solubilization and immunological identification of presynaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors in the rat hippocampus.</a> Neurosci. Lett. 336, 97-100 (2003).</li>
<li>Rebola, N., Pinheiro, P. C., Oliveira, C. R., Malva, J. O., Cunha, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Subcellular+localization+of+adenosine+A%281%29+receptors+in+nerve+terminals+and+synapses+of+the+rat+hippocampus%5BTitle%5D)+AND+%22Brain+Res%22%5BJournal%5D)">Subcellular localization of adenosine A(1) receptors in nerve terminals and synapses of the rat hippocampus.</a> Brain Res. 987, 49-58 (2003).</li>
<li>Rebola, N., Canas, P. M., Oliveira, C. R., Cunha, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Different+synaptic+and+subsynaptic+localization+of+adenosine+A2A+receptors+in+the+hippocampus+and+striatum+of+the+rat%5BTitle%5D)+AND+%22Neuroscience%22%5BJournal%5D)">Different synaptic and subsynaptic localization of adenosine A2A receptors in the hippocampus and striatum of the rat.</a> Neuroscience. 132, 893-903 (2005).</li>
<li>Rodrigues, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dual+Presynaptic+Control+by+ATP+of+Glutamate+Release+via+Facilitatory+P2X1%2C+P2X2%2F3%2C+and+P2X3+and+Inhibitory+P2Y1%2C+P2Y2%2C+and%2For+P2Y4+Receptors+in+the+Rat%5BTitle%5D)+AND+%22J.+Neurosci%22%5BJournal%5D)">Dual Presynaptic Control by ATP of Glutamate Release via Facilitatory P2X1, P2X2/3, and P2X3 and Inhibitory P2Y1, P2Y2, and/or P2Y4 Receptors in the Rat.</a> J. Neurosci. 25, 6286-6295 (2005).</li>
<li>Garção, P., Oliveira, C. R., Cunha, R. A., Agostinho, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Subsynaptic+localization+of+nicotinic+acetylcholine+receptor+subunits%3A+a+comparative+study+in+the+mouse+and+rat+striatum%5BTitle%5D)+AND+%22Neurosci.+Lett%22%5BJournal%5D)">Subsynaptic localization of nicotinic acetylcholine receptor subunits: a comparative study in the mouse and rat striatum.</a> Neurosci. Lett. 566, 106-110 (2014).</li>
<li>Lopes, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+role+of+parkinson%27s+disease-associated+receptor+GPR37+in+the+hippocampus%3A+functional+interplay+with+the+adenosinergic+system%5BTitle%5D)+AND+%22J.+Neurochem%22%5BJournal%5D)">The role of parkinson's disease-associated receptor GPR37 in the hippocampus: functional interplay with the adenosinergic system.</a> J. Neurochem. 134, 135-146 (2015).</li>
<li>Clark, J. D., Gebhart, G. F., Gonder, J. C., Keeling, M. E., Kohn, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Special+Report%3A+The+1996+Guide+for+the+Care+and+Use+of+Laboratory+Animals%5BTitle%5D)+AND+%22ILAR+J%22%5BJournal%5D)">Special Report: The 1996 Guide for the Care and Use of Laboratory Animals.</a> ILAR J. 38, 41-48 (1997).</li>
<li>Rusakov, D. A., Harrison, E., Stewart, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Synapses+in+hippocampus+occupy+only+1-2%25+of+cell+membranes+and+are+spaced+less+than+half-micron+apart%3A+a+quantitative+ultrastructural+analysis+with+discussion+of+physiological+implications%5BTitle%5D)+AND+%22Neuropharmacology%22%5BJournal%5D)">Synapses in hippocampus occupy only 1-2% of cell membranes and are spaced less than half-micron apart: a quantitative ultrastructural analysis with discussion of physiological implications.</a> Neuropharmacology. 37, 513-521 (1998).</li>
<li>Zeng, Z., Su, K., Kyaw, H., Li, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+endothelin+receptor+type-B-like+gene+enriched+in+the+brain%5BTitle%5D)+AND+%22Biochem.+Biophys.+Res.+Commun%22%5BJournal%5D)">A novel endothelin receptor type-B-like gene enriched in the brain.</a> Biochem. Biophys. Res. Commun. 233, 559-567 (1997).</li>
<li>Bodenmuller, H., Schilling, E., Zachmann, B., Schaller, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+neuropeptide+head+activator+loses+its+biological+acitivity+by+dimerization%5BTitle%5D)+AND+%22EMBO+J%22%5BJournal%5D)">The neuropeptide head activator loses its biological acitivity by dimerization.</a> EMBO J. 5, 1825-1829 (1986).</li>
<li>Rezgaoui, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+neuropeptide+head+activator+is+a+high-affinity+ligand+for+the+orphan+G-protein-coupled+receptor+GPR37%5BTitle%5D)+AND+%22J.+Cell+Sci%22%5BJournal%5D)">The neuropeptide head activator is a high-affinity ligand for the orphan G-protein-coupled receptor GPR37.</a> J. Cell Sci. 119, 542-549 (2006).</li>
<li>Gandìa, J., Fernández-Dueñas, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Parkinson%27s+disease-associated+GPR37+receptor-mediated+cytotoxicity+is+controlled+by+its+intracellular+cysteine-rich+domain%5BTitle%5D)+AND+%22J.+Neurochem%22%5BJournal%5D)">The Parkinson's disease-associated GPR37 receptor-mediated cytotoxicity is controlled by its intracellular cysteine-rich domain.</a> J. Neurochem. 125, 362-372 (2013).</li>
<li>Meyer, R. C., Giddens, M. M., Schaefer, S. A., Hall, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((GPR37+and+GPR37L1+are+receptors+for+the+neuroprotective+and+glioprotective+factors+prosaptide+and+prosaposin%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.+S.+A%22%5BJournal%5D)">GPR37 and GPR37L1 are receptors for the neuroprotective and glioprotective factors prosaptide and prosaposin.</a> Proc. Natl. Acad. Sci. U. S. A. 110, 9529-9534 (2013).</li>
<li>Takahashi, R., Imai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pael+receptor%2C+endoplasmic+reticulum+stress%2C+and+Parkinson%27s+disease%5BTitle%5D)+AND+%22J.+Neurol%22%5BJournal%5D)">Pael receptor, endoplasmic reticulum stress, and Parkinson's disease.</a> J. Neurol. 250, 9(2003).</li>
</ol>

The authors have nothing to disclose.

The protocol presented here constitutes a powerful biochemical tool for the study of the subsynaptic distribution of specific proteins within any brain region. However, there are some drawbacks inherent to the technique that deserve to be highlighted here. For instance, one of the main limitations is the relatively large amount of tissue needed to purify a reasonable amount of protein in order to perform the immunoblot analysis of all subsynaptic fractions. This issue might be related to the fact that synapses (i.e.,...

Synaptic transmission relies on the physical integrity of the synapse, a concept that was envisaged as early as 1897 by Foster and Sherrington1. Thus, understanding the distribution of key neurotransmission components (e.g., ion channels, receptors, etc.) is essential to elucidate synaptic function, both in normal and pathological conditions. Electron microscopy (EM) has contributed enormously to the current ultrastructural notion of prototypical central nervous system (CNS) synapses. In such a way, EM has finely established the differences between pre- and postsynaptic densities, which are separated by a cleft of a rather uniform distance (~25 nm)2. Interestingly, the postsynaptic apparatus exhibits a relatively continuous, electron-dense thickening below its plasma membrane, the so-called postsynaptic density, or PSD2. Conversely, at the presynaptic apparatus, a noticeable discontinuous cytomatrix network is arranged just beneath the plasma membrane, which is essential to the alignment and docking of synaptic vesicles to the plasma membrane active zone3. Hence, EM constitutes the golden experimental approach to survey the distribution of proteins within structurally preserved CNS synapses. However, the information provided by electron micrographs is static. Indeed, accumulating evidences show that in vivo synapses are extremely dynamic, thus experiencing dramatic structural changes upon sustained synaptic transmission. In addition, the morphology and composition of synapses can change throughout different CNS regions and upon development, maturation, aging, and the development of neuropathological conditions. Overall, a protocol focused on isolating proteins belonging to different synaptic compartments in physiological conditions represents a valuable tool for a more comprehensive study of synaptic functioning.
Here, we describe this kind of complementary experimental approach, which allows for the preparative biochemical enrichment of the different synaptic membrane compartments-namely, extra-, pre- and postsynaptic membrane domains. This membrane fractionation method, first described by Philips et al. (2001)4, is based on a pH shift that weakens the adhesive interactions occurring within the pre- and postsynaptic apparatus. First, by using mild detergents at pH 6.0, it is possible to discern the adherens junction that holds the pre- and postsynaptic apparatus and that is maintained from the extrasynaptic membrane domain, which is solubilized and thus can be extracted from the synaptic contacts. Subsequently, raising the pH from 6.0 to 8.0 in the presence of mild detergents weakens the strength of the adherens junction that keeps the presynaptic active zone tightly bound to the postsynaptic density. Hence, the presynaptic compartment is solubilized and can be separated from the postsynaptic density, which is mostly preserved because the concentration of detergent used does not promote its solubilization4. Interestingly, the fractionation efficiency, eventually higher than 90%, can be confirmed by different subsynaptic markers: i) synaptosomal-associated protein 25 (SNAP-25), from the presynaptic active zone; ii) synaptophysin, from the extrasynaptic fraction (i.e., outside the active zone and including microsomes); and iii) postsynaptic density protein 95 (PSD-95), from the postsynaptic density. Notably, this brain membrane fractionation method has been used successfully. Accordingly, it has been possible to precisely determine the subsynaptic localization of different receptors, such as alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors5, adenosine A1 receptor (A1R)6, adenosine A2A receptor (A2AR)7, adenosine triphosphate (ATP) P2 receptors8, nicotinic acetylcholine receptor subunits9, and Parkinson&#39;s disease-associated receptor GPR3710. However, a number of limitations may impede the proper assessment of the synaptic distribution of a particular neuronal protein. Thus, in this procedure, we not only fully describe the entire protocol, but we also highlight some critical points to be considered, such as the rather large amount of tissue needed, the low protein yield, and the mandatory requirement to validate the efficiency of each separation before performing the definite experiment.

<table><tbody><tr><td>Sucrose</td><td>Pancreac Qu&amp;iacute;mica SL, Barcelona, Spain</td><td>1,316,211,211</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Pancreac Qu&amp;iacute;mica SL, Barcelona, Spain</td><td>2,112,211,210</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;&amp;middot;6H2O</td><td>Pancreac Qu&amp;iacute;mica SL, Barcelona, Spain</td><td>1,313,961,210</td><td></td></tr><tr><td>Protease inhibitor cocktail Set III</td><td>Millipore, Darmstadt, Germany</td><td>535140</td><td></td></tr><tr><td>Trizma Base</td><td>Sigma, St. Louis, MO, USA</td><td>T1503</td><td></td></tr><tr><td>Tris-HCl</td><td>Pancreac Qu&amp;iacute;mica SL, Barcelona, Spain</td><td>1,236,541,209</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma, St. Louis, MO, USA</td><td>X100</td><td></td></tr><tr><td>SDS</td><td>Sigma, St. Louis, MO, USA</td><td>L3771</td><td></td></tr><tr><td>Glycerol</td><td>Sigma, St. Louis, MO, USA</td><td>G5516</td><td></td></tr><tr><td>Bromophenol Blue</td><td>Pancreac Qu&amp;iacute;mica SL, Barcelona, Spain</td><td>1,311,651,604</td><td></td></tr><tr><td>Dithiothreitol</td><td>Sigma, St. Louis, MO, USA</td><td>D0632</td><td></td></tr><tr><td>Tween 20</td><td>Sigma, St. Louis, MO, USA</td><td>P2287</td><td></td></tr><tr><td>Non fat dry milk</td><td></td><td></td><td></td></tr><tr><td>NaCl</td><td>Pancreac Qu&amp;iacute;mica SL, Barcelona, Spain</td><td>1,216,591,211</td><td></td></tr><tr><td>KCl</td><td>Pancreac Qu&amp;iacute;mica SL, Barcelona, Spain</td><td>1,314,941,210</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Merck</td><td>4873</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Pancreac Qu&amp;iacute;mica SL, Barcelona, Spain</td><td>1,316,781,211</td><td></td></tr><tr><td>Basic 20 pH</td><td>Crison, Alella, Spain</td><td></td><td></td></tr><tr><td>Polytron VDI 12 Adaptable Homogenizer</td><td>VWR, Radnor, PA, USA.</td><td></td><td></td></tr><tr><td>Ultra-Clear Tubes (14x89mm)</td><td>Beckman Coulter, Hospitalet de Llobregat, Barcelona</td><td>344059</td><td>Tubes should be filled almost completely when used to prevent collapsing due to ultracentrifugation.</td></tr><tr><td>Amicon Ultra-15 Centrifugal filters Ultracel -10K</td><td>Merck Millipore, Darmstadt, Germany</td><td>UFC901008</td><td></td></tr><tr><td>Centrifuge 5430R</td><td>Eppendorf, Hamburgo, Germany</td><td></td><td></td></tr><tr><td>Optima L-90K Ultracentrifuge</td><td>Beckman Coulter, Hospitalet de Llobregat, Barcelona</td><td></td><td></td></tr><tr><td>Sonifier 250</td><td>Branson, Danbury, Connecticut</td><td></td><td></td></tr><tr><td>Amersham Imager 600</td><td>GE Healthcare Europe GmbH, Barcelona, Spain</td><td></td><td></td></tr><tr><td>Disposable Glass Pasteur Pippetes 230 mm</td><td>VWR, Radnor, PA, USA</td><td>612-1702</td><td></td></tr><tr><td>Compact Balance EK-610</td><td>A&amp;amp;D, Tokyo, Japan</td><td></td><td></td></tr><tr><td>Pierce&amp;trade; BCA Protein Assay Kit</td><td>Pierce Biotechnology, Rockford, IL, USA</td><td></td><td></td></tr><tr><td>SuperSignal west pico chemiluminescent substrate</td><td>Thermo Fisher Scientific Inc., Waltham, MA, USA</td><td></td><td></td></tr><tr><td>GR 200 Precision Balance</td><td>A&amp;amp;D, Tokyo, Japan</td><td></td><td></td></tr><tr><td>Anti-GPR37</td><td>Homemade antibody anti-GPR37 produced and validated in Francisco Ciruela Laboratory.</td><td></td><td>Primary antibodies used at a final concentration of 0.250 &amp;mu;g/mL</td></tr><tr><td>Anti-SNAP-25, anti-PSD-95, anti-synaptophysin</td><td>Abcam, Cambridge, United Kingdom</td><td></td><td>Primary antibodies diluted 1:10,000</td></tr><tr><td>HRP-conjugated goat anti-mouse IgG</td><td>Thermo Fisher Scientific, Waltham, MA, USA.</td><td></td><td>Secondary antibody diluted 1:10,000</td></tr><tr><td>HRP-conjugated goat anti-rabbit IgG</td><td>Thermo Fisher Scientific, Waltham, MA, USA.</td><td></td><td>Secondary antibody diluted 1:30,000</td></tr></tbody></table>

brain membrane fractionation: an ex vivo approach to assess subsynaptic protein localization

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.

The described methodology has been largely used for the subsynaptic analysis of neuronal proteins in general and for the isolation and biochemical characterization of synaptic receptors5,6,7,8,9 in particular. Interestingly, the representative result displayed here show the usefulness of this experimental procedure for the anal...

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 ...

The overall goal of this procedure is to isolate proteins belonging to different synaptic compartments from mice brain. Describing the distribution of kinetic transmissioncomponents is essential to understand synaptic function both in normal and pathological conditions. Accumulating evidence show that in vivo synapses are extremely dynamic and can undergo dramatic structural changes open sustaining synaptic transmission.Synapses have been classically studied by means of electron microscopy. This membrane fractionation method is based on combination of laser obliteration using mild detergents under different pH conditions followed by split second centrifugation. In brief, mouse brain is removed and its hippocampus is carefully dissected in a nice cold dish seated on crushed ice.The hippocampi are collected into a Potter-Elvehjem glass tube containing ice cold isolation buffer. Subsequently, the collected tissue is homogenized using a homogenizing stirrer. Next the homogenized tissue is mixed with a sucrose calcium solution.Then the tissue extract is overlaid with a concentrated sucrose calcium solution. The samples are then centrifuged. The membrane fraction containing synaptosomes is easily recognized as a dense ring floating within the sucrose solution's interface.Subsequently the isolation of the different sub-synaptic compartments, namely extra, pre, and post synaptic fractions is achieved by combining light solubilization using mild detergents upon differentiation pH condition. Finally, the protein enrichment at the different compartments is validated by immunoblot analysis. The day of the experiment prepare fresh solutions and chill them on ice.Place the brain in a Petri dish seating on crushed ice and containing 10 milliliters of ice cold isolation buffer. Carefully dissect the hippocampus with the help of a pair of forceps. Transfer the hippocampi into a five milliliters Potter-Elvehjem glass tube containing one milliliter of ice-cold isolation buffer.And then homogenize using a homogenizing stirrer. Transfer the homogenized tissue into a 15 milliliters Falcon tube containing the sucrose and calcium solution and mix it slowly by inverting the tube. Place the solution in an ultra-clear centrifuge tube and add on top of each tube 2.5 milliliters of sucrose containing calcium chloride.And calibrate the tubes with isolation buffer solution and centrifuge for three hours at 100, 000 G at four degrees in an ultra centrifuge. Carefully remove the tube from the centrifuge steel support and identify the dense ring within the sucrose interface which contains the synaptosomes. Discard the top mailing layer with the help of a blue tip.With a Pasteur pipette, collect the dense ring containing the synaptosomes fractions and transfer into an ultra-clear centrifuge tube and keep on crushed ice. Dilute the synaptosomes with nine volume of ice cold isolation buffer and centrifuge for 30 minutes at 15, 000 G at four degrees. After centrifugation, remove the tube.Discard the supernatant. A pellet containing the synaptosomes should be seen. Resuspend the pellet with 1.1 milliliters of isolation buffer and keep on crushed ice for the next step.Make fresh solutions with the corresponding pH adjusted at four degrees. Add slowly one milliliter of the resuspended synaptosomal pellet into five milliliters of calcium chloride in a beaker on ice under continuous agitation. Subsequently at five milliliters of concentrated solubilization buffer pH 6.After 50 minutes on ice, transfer the solution into an ultra-clear centrifuge tube and centrifuge at 40, 000 G for 30 minutes at four degrees. After centrifugation, remove the tube. A pellet containing the pre and post-synaptic fraction should be observed.The supernatant containing the extra synaptic fraction is collected in a 15 milliliters concentrating tube. Carefully wash the pellet with the two milliliters solubilization buffer pH six. Avoid disrupting the pellet and discard the buffer.At 10 milliliters of solubilizing buffer pH eight to the pellet, and titrate with the aid of a Pasteur pipette. Incubate a beaker on ice under continuous agitation during 50 minutes. Transfer the solution into an ultra-clear centrifuge tube and centrifuge at 40, 000 G for 30 minutes at four degrees.After centrifugation, remove the tube. A pellet containing the post-synaptic fraction should be observed. The supernatant containing the pre-synaptic fraction is collected in a 15 milliliters concentrating tube.The pellet containing the post-synaptic fraction is resuspended with 200 microliters of STS solution. Sonicate the resuspended pellet. Assess the amount of protein in each fraction by means of a protein determination assay.Take 20 micrograms of protein from each fraction and treat with STS Polyacrylamide Gel Electrophoresis sample buffer. Separate proteins by STS Polyacrylamide Gel Electrophoresis and analyze by immunoblot. GPR37 is another fancy protein global receptor mostly in rodent brain.Implicated in different pathologies such as Parkinson's disease or major depression, its function remains abnormal. Studying the presence of this receptor in synapses can help decipher its role in the central nervous system. Synaptosomes from wild type and GPR37 knockout mice were isolated and subsequently fractionated.The purity of each sub-synaptic compartment was warrantied by the segregation of the respective synaptic markers. The extra synaptic vesicular marker synaptophysin was enriched in this extra synaptic fraction. The BSD 95 protein, a classical post-synaptic density marker was enriched in the corresponding post synaptic fraction.Finally, the purity of pre synaptic fraction was validated by the presence of SNAP 25 a pre synaptic active zone marker. Interestingly while GPR 37 immuno reactivity was more abundant in the extra synaptic fraction, it also showed a significant preference for the post synaptic over the pre-synaptic fraction. Assessing the sub synaptic protein composition is an important challenge in neuroscience.However, this is not an easy task since it is highly regulated by protein protein interaction ribbons. Here we present a brain membrane fractionation protocol that represent a robust procedure to select proteins belonging to different synaptic compartments. In other words, it is described of your chemical technology to carry out protein enrichment from pre synaptic, post synaptic, and extra synaptic compartments.Goodbye and good luck with your fractionation experiments.

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Research

JoVE Journal

Biochemistry

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

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