Prep aration of Synaptic Vesicles from
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
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
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.
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
|FIGURE 1 Flowchart depicting the main steps of the two preparation
methods for synaptic vesicles described in the text. The final
step for both methods is CPG chromatography.
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).
Preparation of Synaptic Vesicles from
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
- Homogenization buffer: 320 mM sucrose, 4 mM HEPES-NaOH, pH 7.3 (HEPES is optional; we
found no difference when the buffer is omitted. Other
buffers such as MES and slightly lower pH are also
- 1 M HEPES-NaOH, pH 7.4
- 40 mM sucrose
- 50 mM sucrose
- 800 mM sucrose
- Glycine buffer: 300 mM glycine, 5 mM
HEPES-KOH, pH 7.4, degassed and filtered through a
- Protease inhibitors: 1 mg/ml pepstatin A in DMSO
and 200 mM PMSF in dry ethanol. Keep stocks at room
temperature. Add 1/1000 volume where indicated.
Note that PMSF is unstable in aqueous solutions and
should not be added to buffers prior to use. The PMSF
stock solution should be prepared fresh every day.
After collecting the brains, all steps are carried out
on ice or at 4°C.
- Decapitate 20 rats (about 2 months old; 180-
200g body weight), remove the brains, avoiding
myelin-rich areas such as corpus callosum or medulla
oblongata, place into 180ml ice-cold homogenization
buffer, and homogenize in several aliquots with a
loose-fitting glass Teflon homogenizer (nine strokes,
900rpm). Add protease inhibitors.
- Centrifuge the homogenate for 10min at
1000gmax (2700rpm, Sorval SS34 rotor), discard the
resulting pellet (P1) containing large cell fragments
and nuclei, and collect the supernatant (S1).
- Centrifuge S1 for 15min at 12,000gmax
(10,000rpm; SS34 rotor); remove the supernatant (S2)
containing small cell fragments such as microsomes or
small myelin fragments and soluble proteins. Wash the
pellet (P2) by carefully resuspending in 120ml homogenization
buffer (pipette, avoiding the dark brown
bottom part of the pellet that consists mainly of
mitochondria) and recentrifuging at 13,000gmax
(11,000rpm, SS34 rotor); discard the supernatant (S2').
The resulting pellet (P2') represents a crude synaptosomal
- To release synaptic vesicles from the synaptosomes,
resuspend P2' in homogenization buffer to
yield a final volume of 12 ml. Transfer this fraction into
a glass-Teflon homogenizer, add 9 volumes (108ml)
ice-cold water and perform three up-and-down
strokes at 2000rpm. Add 1 ml of 1M HEPES-NaOH,
pH 7.4, and protease inhibitors.
- Centrifuge the suspension for 20min at
33,000gmax (16,500rpm, SS34 rotor) to yield the lysate
pellet (LP1) and the lysate supernatant (LS1). Using an
electric pipetter, carefully remove LS1 immediately
after the end of the run without disturbing LP1. It is
crucial that LS1 does not get contaminated even with
traces of membrane fragments from LP1 (rather, leave 1-2ml behind in the tube). Contaminating LS1 with
LP1 is the most common problem, which significantly
reduces the purity of the final vesicle fraction.
- Centrifuge LS1 for 2h at 260,000gmax
(50,000rpm, Beckman 60Ti or comparable rotor).
Discard the supernatant (LS2) and resuspend the pellet
(LP2) in 6 ml of 40mM sucrose utilizing a small, tightfitting
glass-Teflon homogenizer. Extrude the resuspended
sample consecutively through a 23- and a
27-gauge hypodermic needle attached to a 10-ml
syringe (avoid air bubbles).
- Layer the suspension (3-ml aliquots) on top of
a linear sucrose gradient formed from 18.5ml of
800 mM sucrose and 18.5 ml of 50 mM sucrose (prepare
two tubes containing identical gradients in advance)
and centrifuge for 4h at 65,000gav (25,000rpm,
Beckman SW 28 rotor). After the run, a turbid (whiteopaque)
zone is visible in the middle of the gradient
(in the range of 200 to 400 mM sucrose, best seen when
viewed against a black background with light from the
top). Collect these bands with the aid of a glass capillary
connected to a peristaltic pump, yielding a combined
volume of 25-30ml. This fraction represents
synaptic vesicles that are 8- to 10-fold enriched over
the homogenate (Jahn et al., 1985). Note that synaptic
vesicles do not reach isopycnic equilibrium during
this velocity gradient-type centrifugation. Changes of
angular velocity or of the run time will therefore affect
- Equilibrate a CPG-3000 column (180 × 2cm, see
Appendix) with 10 column volumes of glycine buffer
(optimally done overnight before the preparation).
Load the sample on top of the resin and overlay it carefully
with glycine buffer without diluting the sample.
Elute the column with glycine buffer at a flow rate
of 40ml/h, collecting 6- to 8-ml fractions. Monitor
protein effiux at 280 nm. The first peak contains plasma
membranes and some microsomes and is usually
smaller than the second peak containing synaptic vesicles.
If no separation into two clearly distinguishable
peaks is obtained, the column may need to be
repacked. Fractions of the second peak are pooled and
centrifuged for 90min at 260,000gmax (50,000rpm,
Beckman 60Ti rotor). The synaptic vesicle pellet should
have a glassy appearance, being completely transparent
and colorless. Resuspend it in the desired buffer as
in step 6. The suspension is frozen rapidly (e.g., in
liquid nitrogen) and stored at -70°C. Yields are typically
between 2 and 3 mg of protein, based on one of
the commercially available Coomassie blue protein
: 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
B. Preparation of Synaptic Vesicles from
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
The final enrichment factor for synaptic vesicles
purified by this protocol is 15-20 (Hell et al.
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
- Homogenization buffer: 320mM sucrose, degassed
- 700mM sucrose: 700mM sucrose and 10mM HEPES-KOH, pH 7.3
- Resuspension buffer: 320 mM sucrose and 10 mM HEPES-KOH, pH 7.3
- Glycine buffer: 300mM glycine and 5mM HEPES-KOH, pH 7.3, degassed
- Protease inhibitors: 1 mg/ml pepstatin A dissolved in
DMSO and 200mM PMSF in dry ethanol; add
1 / 1000 volume where indicated
After powdering the frozen brains, all steps are
carried out on ice or at 4°C.
- Decapitate 40 rats (2 months old, 180-200 g body
weight); remove the brains, avoiding myelin-rich areas
such as corpus callosum or medulla oblongata, and
freeze immediately in liquid nitrogen. Immediate
shock freezing is essential. In our experience, frozen
brains available from commercial sources are usually
not satisfactory for this reason.
- To create a tissue powder, place the frozen brains
into a porcelain mortar precooled with liquid nitrogen.
Cover them with cheesecloth and break them carefully
using a porcelain pestle. Grind to a fine powder. This
step is crucial for obtaining high yields. After evaporation
of the liquid N2, suspend the powder in 320ml
ice-cold homogenization buffer (magnetic stirrer) and
homogenize with a glass-Teflon homogenizer (eight
strokes, 1000 rpm).
- Centrifuge the homogenate for 10min at
47,000gmax (20,000rpm, Sorval SS-34 rotor). Collect
the supernatant (S1). The pellet (P1) contains large cell
fragments and nuclei, but also some entrapped synaptic
vesicles. To increase the yield, reextract the pellet
with 160ml homogenization buffer by means of one
slow stroke in the glass-Teflon homogenizer followed
by centrifugation as described earlier. The resulting
supernatant (S1') is combined with S1.
- Centrifuge S1 for 40min at 120,000gmax
(32,000rpm, Beckman 45Ti rotor). Using an electric
pipetter, collect the supernatant (S2) carefully without
disturbing the pellet (P2). It is crucial that S2 is not contaminated
with membrane fragments from the soft
pellet P2. S2 should be clear with a reddish color.
If it is turbid, it should be recentrifuged using the
same conditions to remove contaminating membrane
- To sediment synaptic vesicles through a sucrose
cushion, fill 25-ml centrifuge tubes appropriate for a
Beckman 60Ti rotor with 20ml S2. Form the sucrose
cushion by pumping 5.5 ml of 700mM sucrose underneath
S2 using a peristaltic pump and a glass capillary.
Centrifuge for 2h at 260,000gmax (50,000rpm,
Beckman 60Ti rotor). Remove the supernatant S3 and
resuspend the pellet P3 in 6-10 ml resuspension buffer
with a small, tight-fitting glass-Teflon homogenizer.
Extrude the resuspended sample consecutively
through a 23- and a 27-gauge hypodermic needle
attached to a 10-ml syringe (avoid air bubbles). This
sample represents a crude synaptic vesicles fraction.
Clear the suspension by a short spin (10min) at
35,000gmax (17,000 rpm, SS34) before loading onto the
- Equilibrate a CPG column (see Appendix) with
10 column volumes of glycine buffer. Load the sample
on top of the resin and overlay carefully with glycine
buffer without disturbing the sample. A column of size
85 × 1.6 cm has a maximal capacity of 15 mg of protein,
requiring several consecutive runs if all material is to
be chromatographed. Elute the column with glycine
buffer at a flow rate of 80ml/h, collecting 2-ml fractions.
Follow the elution of protein with a UV detector
at 280nm. The first peak, containing plasma membranes
and microsomes, is usually larger than the
second peak, containing synaptic vesicles. The two
peaks are typically not completely separated in this
protocol. The shoulder frequently observed at the end
of the second peak represents soluble protein. Pool the
fractions of the second peak and centrifuge for 2h at
260,000gmax (50,000rpm; 60Ti rotor). Resuspend the
synaptic vesicle pellet in the desired buffer as
described in step 5.
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.
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
7.4, and centrifuge for 2 h at 50,000rpm, 260,000g
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,
|FIGURE 2 Electron micrograph showing a synaptic vesicle fraction
purified by the procedure described in Section IIIB (negative
staining). (Inset) Magnification of a field following immunogold
labeling for the synaptic vesicle protein synaptophysin. For
methods, see Jahn and Maycox (1988). Bar: 200nm. Electron micrographs
Dr. Peter R. Maycox (London, UK).
The authors thank Dr. Duane D. Hall for preparation
of Fig. 1.
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