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

Summary

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

Abstract

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.

Introduction

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 uni....

Protocol

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 °C temperature, and 66% humidity).

1. Obtaining Mouse Brain Synaptosomes Using a Discontinuous Sucrose Gradient

Results

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 receptors^5,6,7,8,9 in particular. Interestingly, the representative result displayed here show the usefulness of this experimental procedure for the anal.......

Discussion

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.,.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Sucrose	Pancreac Química SL, Barcelona, Spain	1,316,211,211
CaCl2	Pancreac Química SL, Barcelona, Spain	2,112,211,210
MgCl2·6H2O	Pancreac Química SL, Barcelona, Spain	1,313,961,210
Protease inhibitor cocktail Set III	Millipore, Darmstadt, Germany	535140
Trizma Base	Sigma, St. Louis, MO, USA	T1503
Tris-HCl	Pancreac Química SL, Barcelona, Spain	1,236,541,209
Triton X-100	Sigma, St. Louis, MO, USA	X100
SDS	Sigma, St. Louis, MO, USA	L3771
Glycerol	Sigma, St. Louis, MO, USA	G5516
Bromophenol Blue	Pancreac Química SL, Barcelona, Spain	1,311,651,604
Dithiothreitol	Sigma, St. Louis, MO, USA	D0632
Tween 20	Sigma, St. Louis, MO, USA	P2287
Non fat dry milk
NaCl	Pancreac Química SL, Barcelona, Spain	1,216,591,211
KCl	Pancreac Química SL, Barcelona, Spain	1,314,941,210
KH2PO4	Merck	4873
Na2HPO4	Pancreac Química SL, Barcelona, Spain	1,316,781,211
Basic 20 pH	Crison, Alella, Spain
Polytron VDI 12 Adaptable Homogenizer	VWR, Radnor, PA, USA.
Ultra-Clear Tubes (14x89mm)	Beckman Coulter, Hospitalet de Llobregat, Barcelona	344059	Tubes should be filled almost completely when used to prevent collapsing due to ultracentrifugation.
Amicon Ultra-15 Centrifugal filters Ultracel -10K	Merck Millipore, Darmstadt, Germany	UFC901008
Centrifuge 5430R	Eppendorf, Hamburgo, Germany
Optima L-90K Ultracentrifuge	Beckman Coulter, Hospitalet de Llobregat, Barcelona
Sonifier 250	Branson, Danbury, Connecticut
Amersham Imager 600	GE Healthcare Europe GmbH, Barcelona, Spain
Disposable Glass Pasteur Pippetes 230 mm	VWR, Radnor, PA, USA	612-1702
Compact Balance EK-610	A&D, Tokyo, Japan
Pierce™ BCA Protein Assay Kit	Pierce Biotechnology, Rockford, IL, USA
SuperSignal west pico chemiluminescent substrate	Thermo Fisher Scientific Inc., Waltham, MA, USA
GR 200 Precision Balance	A&D, Tokyo, Japan
Anti-GPR37	Homemade antibody anti-GPR37 produced and validated in Francisco Ciruela Laboratory.		Primary antibodies used at a final concentration of 0.250 μg/mL
Anti-SNAP-25, anti-PSD-95, anti-synaptophysin	Abcam, Cambridge, United Kingdom		Primary antibodies diluted 1:10,000
HRP-conjugated goat anti-mouse IgG	Thermo Fisher Scientific, Waltham, MA, USA.		Secondary antibody diluted 1:10,000
HRP-conjugated goat anti-rabbit IgG	Thermo Fisher Scientific, Waltham, MA, USA.		Secondary antibody diluted 1:30,000