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...

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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 border="0" cellpadding="0" cellspacing="0" width="704">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Sucrose</td>
			<td style="width:249px;">Pancreac Qu&#237;mica SL, Barcelona, Spain</td>
			<td style="width:160px;">1,316,211,211</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">CaCl2</td>
			<td style="width:249px;">Pancreac Qu&#237;mica SL, Barcelona, Spain</td>
			<td style="width:160px;">2,112,211,210</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">MgCl2&#183;6H2O</td>
			<td style="width:249px;">Pancreac Qu&#237;mica SL, Barcelona, Spain</td>
			<td style="width:160px;">1,313,961,210</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:128px;">Protease inhibitor cocktail Set III</td>
			<td style="width:249px;">Millipore, Darmstadt, Germany</td>
			<td style="width:160px;">535140</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Trizma Base</td>
			<td style="width:249px;">Sigma, St. Louis, MO, USA</td>
			<td style="width:160px;">T1503</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Tris-HCl</td>
			<td style="width:249px;">Pancreac Qu&#237;mica SL, Barcelona, Spain</td>
			<td style="width:160px;">1,236,541,209</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Triton X-100</td>
			<td style="width:249px;">Sigma, St. Louis, MO, USA</td>
			<td style="width:160px;">X100</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">SDS</td>
			<td style="width:249px;">Sigma, St. Louis, MO, USA</td>
			<td style="width:160px;">L3771</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Glycerol</td>
			<td style="width:249px;">Sigma, St. Louis, MO, USA</td>
			<td style="width:160px;">G5516</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Bromophenol Blue</td>
			<td style="width:249px;">Pancreac Qu&#237;mica SL, Barcelona, Spain</td>
			<td style="width:160px;">1,311,651,604</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Dithiothreitol</td>
			<td style="width:249px;">Sigma, St. Louis, MO, USA</td>
			<td style="width:160px;">D0632</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Tween 20</td>
			<td style="width:249px;">Sigma, St. Louis, MO, USA</td>
			<td style="width:160px;">P2287</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Non fat dry milk</td>
			<td style="width:249px;"></td>
			<td style="width:160px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">NaCl</td>
			<td style="width:249px;">Pancreac Qu&#237;mica SL, Barcelona, Spain</td>
			<td style="width:160px;">1,216,591,211</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">KCl</td>
			<td style="width:249px;">Pancreac Qu&#237;mica SL, Barcelona, Spain</td>
			<td style="width:160px;">1,314,941,210</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">KH2PO4</td>
			<td style="width:249px;">Merck</td>
			<td style="width:160px;">4873</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Na2HPO4</td>
			<td style="width:249px;">Pancreac Qu&#237;mica SL, Barcelona, Spain</td>
			<td style="width:160px;">1,316,781,211</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Basic 20 pH</td>
			<td style="width:249px;">Crison, Alella, Spain</td>
			<td style="width:160px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:128px;">Polytron VDI 12 Adaptable Homogenizer</td>
			<td style="width:249px;">VWR, Radnor, PA, USA.</td>
			<td style="width:160px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="65">
			<td height="65" style="height:65px;width:128px;">Ultra-Clear Tubes (14x89mm)</td>
			<td style="width:249px;">Beckman Coulter, Hospitalet de Llobregat, Barcelona</td>
			<td style="width:160px;">344059</td>
			<td style="width:167px;">Tubes should be filled almost completely when used to prevent collapsing due to ultracentrifugation.</td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:128px;">Amicon Ultra-15 Centrifugal filters Ultracel -10K</td>
			<td style="width:249px;">Merck Millipore, Darmstadt, Germany</td>
			<td style="width:160px;">UFC901008</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Centrifuge 5430R</td>
			<td style="width:249px;">Eppendorf, Hamburgo, Germany</td>
			<td style="width:160px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:128px;">Optima L-90K Ultracentrifuge</td>
			<td style="width:249px;">Beckman Coulter, Hospitalet de Llobregat, Barcelona</td>
			<td style="width:160px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Sonifier 250</td>
			<td style="width:249px;">Branson, Danbury, Connecticut</td>
			<td style="width:160px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Amersham Imager 600</td>
			<td style="width:249px;">GE Healthcare Europe GmbH, Barcelona, Spain</td>
			<td style="width:160px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:128px;">Disposable Glass Pasteur Pippetes 230 mm</td>
			<td style="width:249px;">VWR, Radnor, PA, USA</td>
			<td style="width:160px;">612-1702</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:128px;">Compact Balance EK-610</td>
			<td style="width:249px;">A&#38;D, Tokyo, Japan</td>
			<td style="width:160px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:128px;">Pierce&#8482; BCA Protein Assay Kit</td>
			<td style="width:249px;">Pierce Biotechnology, Rockford, IL, USA</td>
			<td style="width:160px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:128px;">SuperSignal west pico chemiluminescent substrate</td>
			<td style="width:249px;">Thermo Fisher Scientific Inc., Waltham, MA, USA</td>
			<td style="width:160px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:128px;">GR 200 Precision Balance</td>
			<td style="width:249px;">A&#38;D, Tokyo, Japan</td>
			<td style="width:160px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:128px;">Anti-GPR37</td>
			<td style="width:249px;">Homemade antibody anti-GPR37 produced and validated in Francisco Ciruela Laboratory.</td>
			<td style="width:160px;"></td>
			<td style="width:167px;">Primary antibodies used at a final concentration of 0.250ug/ml</td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:128px;">Anti-SNAP-25, anti-PSD-95, anti-synaptophysin</td>
			<td style="width:249px;">Abcam, Cambridge, United Kingdom</td>
			<td style="width:160px;"></td>
			<td style="width:167px;">Primary antibodies diluted 1:10000</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:128px;">HRP-conjugated goat anti-mouse IgG</td>
			<td style="width:249px;">Thermo Fisher Scientific, Waltham, MA, USA.</td>
			<td style="width:160px;"></td>
			<td style="width:167px;">Secondary antibody diluted 1:10000</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:128px;">HRP-conjugated goat anti-rabbit IgG</td>
			<td style="width:249px;">Thermo Fisher Scientific, Waltham, MA, USA.</td>
			<td style="width:160px;"></td>
			<td style="width:167px;">Secondary antibody diluted 1:30000</td>
		</tr>
	</tbody>
</table>

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...

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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.

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 ...

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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Biochemistry

10.2K visualizações.  Universitat de Barcelona, L'Hospitalet de Llobregat. 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 micros...