Prep aration of Synaptic Vesicles from Mammalian Brain
Synaptic vesicles are secretory organelles that store neurotransmitters in presynaptic nerve endings. When an action potential arrives in the nerve terminal, the plasma membrane is depolarized, leading to the opening of voltage-gated Ca2+ channels. The rise in intracellular Ca2+ concentration leads to exocytosis of synaptic vesicles within a time interval that can be as short as 200 µs (reviewed by Südhof, 1995).
Synaptic vesicles possess several remarkable properties that distinguish them from most other organelles involved in membrane traffic. First, they are very abundant in brain tissue. Model calculations show that an average neuron contains approximately 106 synaptic vesicles, with a total of around 1017 in the human central nervous system (Jahn and Sfidhof, 1993). Approximately 5% of the protein in the brain is contributed by synaptic vesicles; thus, about a 20-fold enrichment from homogenate is sufficient to obtain a pure preparation. Second, synaptic vesicles are highly homogeneous in size and shape and, in addition, are smaller than most other organelles, with an average diameter of only 50nm. Therefore, size-fractionation techniques can be applied for the isolation of synaptic vesicles. Third, synaptic vesicles do not contain a matrix of soluble proteins (Jahn and Sfidhof, 1993), as they recycle many times in the nerve terminal and thus can only be reloaded with nonpeptide transmitters by means of specific transport systems.
The study of synaptic vesicles has been facilitated by recent advances in understanding their protein composition. To date, more than half a dozen protein families have been shown to be localized specifically on the membrane of synaptic vesicles. With the exception of some variation due to isoforms, most of these proteins are residents of all synaptic vesicles irrespective of their neurotransmitter content or of the location of the neuron. These include synapsins, synaptophysins, synaptotagmins, SV2s, synaptobrevins/ VAMPs, rabs, cysteine string proteins, synaptogyrin, and the subunits of the Vacuolar proton pump (for a review, see Sfidhof, 1995). Rapid progress during the last few years has clarified the function of many of these proteins in membrane traffic. Synaptic vesicles are thus presently regarded as the best-characterized "model" trafficking organelle of eukaryotic cells (Sfidhof, 1995). Antibodies against synaptophysin and synaptobrevin/VAMP are available commercially from several sources and may be used as probes to assess synaptic vesicle purity. In addition, synaptic vesicles contain neurotransmitter transporters that are specific for neurons exhibiting the corresponding neurotransmitter phenotype. Transporters for the main neurotransmitters have been cloned and sequenced (Reimer et al., 2001).
Purification protocols for synaptic vesicles can be divided into three groups. The first group involves the preparation of isolated nerve terminals (synaptosomes) by differential centrifugation. The synaptosomes are subsequently lysed in order to release the synaptic vesicles. An advantage of this procedure is that small membrane fragments generated during homogenization are removed prior to vesicle extraction as synaptosomes sediment at lower g forces than these fragments. The first protocol described here belongs in this category. These protocols are laborious and result in relatively low yields, but the resulting synaptic vesicles preparations are of high purity. In the second group of protocols, synaptic vesicles are purified directly from homogenate, without prior isolation of synaptosomes. In order to obtain high yields, initial homogenization is harsh in order to break up as many nerve terminals as possible, e.g., by freeze-powder homogenization, as described in the second procedure. In both cases, a combination of differential centrifugation, rate-zonal density gradient centrifugation or isopycnic density gradient centrifugation, and size-exclusion chromatography is employed for purification (Fig. 1). The third group involves immunoisolation using antibodies specific for synaptic vesicle proteins (see, e.g., Burger et al., 1989, Walch-Solimena et al., 1993). These procedures allow for the rapid isolation of small quantities of highly pure organelles from brain homogenates. However, they require access to large amounts of specific antibodies (preferably monoclonal antibodies) and are therefore not further discussed here.
II. MATERIALS AND INSTRUMENTATION
The following chemicals are used: HEPES (Research Products International, #H75030), sucrose (Sigma, # S-9378), glycine (Bio-Rad, 161-0718), phenylmethylsulfonyl fluoride (PMSF; ICN, #195381), pepstatin A (ICN, #195368), dimethyl sulfoxide (DMSO, Sigma, #D-8779), and controlled pore glass beads (CPG Inc., see Appendix). Note that the standard reagents can also be obtained from other sources.
The following instrumentation is required: loosefitting, motor-driven glass-Teflon homogenizer (Braun, Melsungen, Germany), cooled centrifuge [Sorvall RC5 (DuPont) or comparable, SS34 rotor], ultracentrifuge with fixed angle and swing-out rotors [Beckman L80 (Beckman Instruments) or comparable, Ti70 or Ti50.2 rotor, SW-28 rotor, Ti 45 rotor] and corresponding tubes, equipment for column chromatography (peristaltic pump, UV monitor, fraction collector), gradient mixer for forming continuous sucrose gradients, filtration device for the filtration of buffers using 0.45-µ membranes (Millipore), and glass columns (see Appendix).
A. Preparation of Synaptic Vesicles from Synaptosomes
In this protocol (Nagy et al., 1976; Huttner et al., 1983), a crude synaptosomal fraction (P2) is first isolated by differential centrifugation. The synaptosomes are then lysed by osmotic shock and synaptic vesicles are released into the medium. After removal of synaptosomal fragments and large membranes, synaptic vesicles are sedimented by high-speed centrifugation. The resulting pellet, already five- to sixfold enriched in synaptic vesicles, is then purified further by sucrose velocity-density gradient centrifugation and sizeexclusion chromatography on controlled pore glass beads (CPG). This procedure is the standard method for obtaining synaptic vesicles of the highest purity, with less than 5% contamination as judged by electron microscopy and biochemical analysis. This preparation does contain, however, endosomes derived from nerve terminals and decoated coated vesicles that lost their clathrin but retained adaptors. The degree of this contamination is probably minor but cannot be quantified easily.
After collecting the brains, all steps are carried out on ice or at 4°C.
Note: Size-exclusion chromatography on glycerylcoated CPG beads or on Sephacryl S-1000 is omitted in many protocols and, if applied, is the last step of the procedure. Both resins have a relatively low capacity, do not tolerate overloading, and require some experience in their use. Sephacryl S-1000 has higher separation capacity than CPG per gel volume, but the columns have low flow rates, do not tolerate increased pressure, and have a tendency to adsorb proteins and membrane particles, particularly during the first few separation runs in the life of the column. CPG columns are more difficult to set up and tolerate less material. However, glass beads are noncompressible and allow high flow rates, shortening separation times substantially. The experimenter who does not shy away from the effort to set up a large CPG column is rewarded with highly reliable results for many runs and exceptionally clean synaptic vesicle preparations. We utilized a CPG-3000 column (3 × 180 cm) continuously for 10 years for more than 200 synaptic vesicles preparations, with only a few repackings required, usually caused by experimental error (running dry). Column profiles and synaptic vesicle purity were highly reproducible.
B. Preparation of Synaptic Vesicles from Frozen Brain
This procedure starts with a harsh homogenization of frozen brains to efficiently break up the nerve terminals, thus releasing synaptic vesicles. Frozen brains are ground in a precooled mortar to yield a fine powder. This treatment does not affect the function or integrity of the small synaptic vesicles, but larger membrane structures are ruptured. After resuspending the tissue powder in sucrose solution, most of the cell fragments are removed by centrifugation with low and intermediate angular velocities, leaving synaptic vesicles in the supernatant. Synaptic vesicles are then sedimented at high speed through a cushion of 0.7M sucrose, removing soluble proteins and membrane contaminants of lower buoyant density (mostly myelin). Synaptic vesicles are five- to sixfold enriched in the pellet and can be purified further by CPG chromatography.
The final enrichment factor for synaptic vesicles purified by this protocol is 15-20 (Hell et al., 1988), somewhat lower than in the previous method. However, there are several advantages. First, the tissue can be collected before the experiment and can be stored in liquid nitrogen for more than 1 year, allowing more efficient use of experimental animals. Second, the yield is severalfold higher under optimal conditions than the yield of the preparation from synaptosomes. Third, the procedure is faster, requiring only 12 h for completion, thereby allowing for higher activactivity of various synaptic vesicle functions such as neurotransmitter uptake.
After powdering the frozen brains, all steps are carried out on ice or at 4°C.
Scaling up or down is feasible but it should be kept in mind that changing rotors or using half-filled centrifuge tubes may affect yield and purity significantly and adversely. Contamination by other subcellular fractions (e.g., plasma membranes, mitochondria, endoplasmic reticulum) can be monitored conveniently by assaying for marker enzymes (Hell et al., 1988). In parallel to a decrease of these marker enzymes, proteins specific for synaptic vesicles, namely synaptophysin (p38), for which antibodies are available commercially (e.g., from Boehringer, Mannheim, Germany), should be enriched about 20- to 25-fold over homogenate (Jahn et al., 1985), best quantitated by immunoblotting.
Synaptobrevin is less reliable for quantification by SDS-PAGE/immunoblotting as histones present in the homogenate migrate alongside synaptobrevin/VAMP in nuclei-containing fractions (homogenate) and interfere with the signal, resulting in overestimation of the enrichment factor. The protein profile of the synaptic vesicles preparation as observed after SDS-PAGE exhibits a characteristic pattern (Huttner et al., 1983; Hell et al., 1988), with the prominent membrane proteins synaptobrevin/VAMP, synaptophysin (p38), synaptotagmin (p65), and synapsin I being clearly visible. Synaptic vesicle preparations contain various amounts of soluble proteins with affinity for membranes such as glyceraldehyde phosphate dehydrogenase, aldolase, actin, and tubulin. These proteins may be partially removed by a salt wash (resuspend synaptic vesicles in 160 mM KCl, 10 mM HEPES-KOH, pH 7.4, and centrifuge for 2 h at 50,000rpm, 260,000gmax). However, this treatment also removes synaptic vesicle protein synapsin I.
The morphology of the synaptic vesicles fraction can be studied using electron microscopy (Fig. 2). Membranes can be visualized easily, e.g., by negative staining (Hell et al., 1988). Synaptic vesicle membranes are identified by their very uniform appearance (small vesicular profiles of approximately 50nm diameter). Confirmation can also be obtained by immunogold labeling for the vesicle protein synaptophysin, which can be carried out conveniently on a single day when combined with negative staining (Jahn and Maycox, 1988).
The authors thank Dr. Duane D. Hall for preparation of Fig. 1.
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