Rapid Preparation of Synaptosomes from Mammalian Brain Using Nontoxic Isoosmotic Gradient Material (Percoll)

Journol of Neurocheniir!~ Raven Press, New York G 1984 International Society for Neurochemistry

Rapid Preparation of Synaptosomes from Mammalian Brain Using Nontoxic Isoosmotic Gradient

Material (Percoll)

Agnes Nagy and Antonio V. Delgado-Escueta

Department of Neurology. Reed Neurological Research Center, UCLA Center for Health Sciences, Los Angeles, California, U.S.A.

Abstract: A new procedure is described for the isolation of synaptosomes from various parts of mammalian brain. This method utilizes an isoosmotic Percoll@/sucrose discontinuous gradient and has some advantages over the traditionally used synaptosomal isolation techniques: (1) it is possible to prepare suitable gradients while retaining isoosmolarity; (2) the time of the preparation is remarkably short (approximately 1 h); (3) if necessary, the gradient material can be easily removed from the samples. Intact synaptosomes were recovered from the 10%/16% (voUvol) Percoll interphase. The fractions were identified and characterized by electron microscopy and by several

Conditions utilized to prepare synaptosomes and the duration of each purification step greatly affect the metabolic state of subcellular fractions. Most of the methods used at present are based on the "clas- sical" sucrose density gradient techniques of Gray and Whittaker (1962) and De Robertis et al. (1962) (for reviews see Whittaker, 1969; De Belleroche and Bradford, 1973). These techniques usually involve lengthy high-speed centrifugations (3-4 h), and the hyperosmolarity of the sucrose solutions required for the appropriate density gradients may be phys- iologically harmful to the synaptosomes (Whittaker, 1969; Day et al., 1971).

The introduction of FicolVsucrose gradients (Ku- rokawa et al., 1965; Abdel-Latif, 1966; Autilio et al., 1968; Cotman and Matthews, 1971) eliminated the problem of hyperosmolarity, thereby increasing the metabolic activity of purified synaptosomes (Verity, 1972). The use of a Ficoll flotation tech-

biochemical markers for synaptosomes and other subcellular organelles. The homogeneity of the preparations is comparable to or better than that of synaptosomes prepared by the conventional methods. This procedure has been successfully used for the isolation of synaptosomes from very small tissue samples of various experimental animals and human brain. Key Words: Synaptosomes- Mitochondria-Isolation-Percoll density gradient- Cerebral cortex. Nagy A. and Delgado-Escueta A. V. Rapid preparation of synaptosomes from mammalian brain using nontoxic isoosmotic gradient material (Per- coll). J . Neurochem. 43, 1114-1123 (1984).

nique remarkably reduced the processing time (Booth and Clark, 1978). However, Ficoll adheres to biological membranes, compromising biochemical studies.

Colloid silica sols (Ludox) were introduced as gradient media (Mateyko and Kopac, 1963) but the silica sols were found to be toxic to living cells. Addition of polymers such as polyvinylpirrolidone (PVP) and polyethylene glycol (PEG) increased the stability of the colloids and decreased their toxic effects (Lagercrantz and Pertoft, 1972). However, the large excess of free polymer in the solutions increased the osmolarity and viscosity of the media and was difficult to remove from the biological samples.

The development of Percoll (PVP-coated colloidal silica) overcame these problems. This gradient material has been successfully used for the separation of various cell types and subcellular particles

Received October 31, 1983; accepted March 21, 1984. Address correspondence and reprint requests to Agnes Nagy,

Department of Neurology, Reed Neurological Research Center, UCLA Center for Health Sciences, 710 Westwood Plaza, LAX Angeles, CA 90024, U.S.A.

Abbreviurions used: CNPase, 2', 3'-Cyclic nucleotide 3'-phos-

phohydrolase; HEPES, N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic acid; 5-HT, 5-Hydroxytryptamine; LDH, Lactate dehydrogenase; NA, Noradrenaline; NCR, NADPH-cytochrome c-reductase; PEG, Polyethylene glycol; PVP, Polyvinyl- pirrolidone; SDH, Succinic dehydrogenase; SIP, Stock solution of Isoosmotic Percoll.

1114

ISOLATION OF S YNAPTOSOMES ON PERCOLL GRADIENT 1115

(Wolff, 1975; Cooper and Perry, 1980; Pertoft and Laurent, 1982). in this paper w e report the use of a discontinuous Percoll gradient for the isolation of intact nerve endings from mammalian brain tissues. The advantages of this technique include substan- tially shortened preparation times, maintenance of isoosmolarity, use of a n innocuous material to yield metabolically active synaptosomes, and very good purity.

A preliminary account of this procedure has been presented (Nagy and Delgado-Escueta, 1983).

MATERIALS AND METHODS Special reagents were obtained from Sigma Chemical,

St. Louis, except Percollm density gradient material which is a product of Pharmacia Fine Chemicals AB, Uppsala, Sweden.

Primary subcellular fractionation Male rats of the Sprague-Dawley strain (80- I00 g) and

adult mongolian gerbils of either sex (65-95 g) from the WJL/UC strain (UCLA breeding colony, kindly provided through Dr. J. Bajorek) were used throughout the study. Subcellular fractions were obtained from the gray material of brain cortex and from the hippocampus of the experimental animals. Human brain specimens were obtained during anterior temporal lobectomies, provided by the UCLA Center for the Health Sciences Neuropa- thology Laboratories. Isolation of subcellular particles has been exclusively performed from fresh, nonfrozen brain specimens. All preparatory steps were carried out at 0-4°C.

The scheme for preparing the synaptosomal fraction from brain samples is summarized in Fig. 1 .

Brain samples were gently homogenized (16 up-and- down strokes with a I-min cooling period after first 8) in 10 times volume (or in case of small tissue samples in 20 times volume) of Medium I(0.32 M sucrose; 5 mM N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES), pH 7.5; 0.1 mM EDTA) with a Teflon-glass homogenizer (stated radial clearance: 0.125 mm) at approximately 800 rpm. The crude homogenate was centrifuged at 1 ,OOO x g,, for 10 min to give a pellet (Pi) containing nuclear and cell debris, and the low-speed supernatant (Si). No further washings of PI were carried out.

S, supernatant was centrifuged at 12,000 x g,, for 20 min to produce the crude mitochondrial pellet (P,). After the supernatant (S,) was decanted, the crude mitochondrial pellet was resusgended in 3 ml of isolation medium (Medium I) per gram of original wet tissue (the final protein concentration of P, was approximately 6-8 mg/ml) by gentle hand-homogenization in a small-volume Teflon- glass homogenizer (clearance: 0.2 mm).

With small tissue samples, the S, fraction (up to 1.0 ml volume) was directly layered onto the Percoll gradient.

Preparation of Percoll gradient and synaptosomes All Percoll solutions were prepared just prior to use.

The Stock solution of Zsoosmotic Percoll (SIP) was made by addition of nine parts (voUv01) of original Percoll solution (loo%, vol/vol) to one part (volhol) of 2.5 M sucrose. The published density of the 90% stock isoosmotic PercolUsucrose solution is 1.149 g/ml (Pharmacia). Lower

Crude mitochondrial pellet (P,)

(reauapended in Isolation msdium)

-tM Dilution with 8.5% Percoll medium

isoosmotic I H 10.0% Percoll in

sUc’o’e 1 SYNAPTOSOYES

16.0% 4.0 nrl

W

15,- saw x 20 m*L

FIG. 1. Flow diagram of the procedure followed in the isolation of synaptosomes on an isoosmotic Percoll step gradient. Time of centrifugation from beginning of acceleration to beginning of deceleration. Isolation medium and Percoll medium are defined in Materials and Methods.

density media were prepared by appropriate dilution of SIP with Medium I1 (0.25 M sucrose; 5 mM HEPES, pH 7.2, 0.1 mM EDTA). Note that Percoll has a pH of about 9.0; therefore, the pH of the gradient solutions must be adjusted to pH 7.5. For more details about the charac- teristics of Percoll see the technical booklet, ‘‘PercolP; Methodology and Applications” by Pharmacia.

Percoll step gradients were produced by overlayering 4 ml of 16% with 4 ml of 10% (voUvo1) Percoll solution, and the gradients were kept on ice until use. The resus- pended P, (or S,) fraction (0.5 ml) was then diluted with eight volumes (4 ml) of 8.5% Percoll/sucrose isoosmotic medium. The final concentration of Percoll was 7.5%. This suspension (4.5 ml) was layered onto the 10%/16% Percoll gradient and centrifuged at 15,000 x gavfor 20 min (wz? setting was 1.59 X lo9 radians%). Centrifuga- tion can be carried out either in an ultracentrifuge swinging bucket rotor (e.g., Beckman SW 27.1) or in a fixed-angle rotor of a superspeed centrifuge (e.g., Sorvall SM-24) with hard-wall polycarbonate tubes.

Synaptosomes (banded at the 10%/16% Percoll inter- face) and other fractions were collected from the gradients with a wide-tip Pasteur pipette and analyzed without further purification. When it was necessary, synaptosomes were separated from coated silica particles either by repeated washing of the samples with isolation medium (two or three times at 15,000 X g,, for 20 min), or by a single high-speed centrifugation step ( 145,000 x ga,for 60 min, d t = 6.32 X loio) in an angle-head rotor (e.g., Beckman type Ti-50).

Synaptic plasma membranes. Synaptic plasma membranes were prepared according to the method of Gurd et al. (1974).

Mitochondria. For preparation of mitochondria we essentially followed the procedure used for isolating synaptosomes. However, the density gradient consisted of 8.5%. lo%, and 20% Percoll layers. By increasing the concentration of the bottom Percoll layer from 16% to 20%, the amount of synaptosomal “contamination” that pelleted together with mitochondria was considerably reduced. Pellets at the bottom of the gradients were resus- pended in isoosmotic medium and used as the mitochondrial fraction without any further purification.

1. NPirrorirPm., Voi . 43, N o . 4. I984

1116 A. NAGY AND A . V . DELGADO-ESCUETA

Microsomas. For preparation of microsomes, the 12.OOO x pa, supernatant ( S z ) of the primary fractionation was centrifuged for another 30 min at 18,OOO x gav. This supernatant was taken and further centrifuged at 100,OOO x, g,,. for 60 min. The resulting pellet was used as the microsomal fraction for marker enzyme assays.

Solubl~ proteins. The soluble protein reference fraction was the supernatant remaining after pelleting the microsomes.

Enzyme assays The following marker enzymes were assayed in a

Beckman Acta CII Spectrophotometer: lactate dehydrogenase (LDH) (EC I . 1.1.27; Johnson. 1960): NADPH:cytochrome c oxidoreductase (NCR) (EC 1.6.2.4; Omura and Takesue, 1970); the rotenone-insensitive NADH:cytochrome c oxidoreductase (EC 1.6.99.3; Duncan and Machler, 1966) in the presence of 2 p M rotenone; cytochrome c oxidase (EC 1.9.3.1; Cooperstein and Lazaron, 1951); succinic dehydrogenase (SDH) (EC 1.3.1.6; Earl and Korner, 1%5); 2‘, 3‘-cyclic nucleotide 3’-phosphohydrolase (CNPase) (EC 3.1.4.16; Olafson et al., 1969); and Na+, K+-ATPase (EC 3.6.1.4; Hosie, 1965; Lanzetta et al., 1979).

Samples (5-25 pl) were taken directly from the gradients, and thus all enzyme assays were camed out in the presence of 0.1-0.4% Percoll. Control experiments showed that this amount of Percoll did not interfere with any of the above listed enzyme assays. Nevertheless, the proper amount of Percoll was always added to the incu- bation mixtures of reference fractions.

Note that all enzyme reaction media were made isotonic by the addition of 250 mM sucrose.

Other analytical procedures ATP was assayed by the Stanley and Williams (1969)

firefly tail luciferin-luciferase method as modified by Dowdall et al. (1974). The assay was sensitive over the range 0.04- 1 .OO nmol. Catecholamines were determined by a combined technique of Hathaway et al. (1967) and Chang (1964). 5-Hydroxytryptamine (5-HT, serotonin) was detected according to Snyder et al. (1965).

Protein was determined by the technique of Peterson (1977), or when Percoll was present, by the Coomassie Blue method of Bradford (1976).

Electron microscopy Fractions obtained from the density gradients were

fixed by the slow addition of ice-cold buffered (pH 7.4) isoosmotic (by the addition of sucrose) glutaraldehyde solution (end concentrations: 4%). After approximately 30 min, subcellular particles were separated from Percoll particles by high-speed centrifugation (145,000 x g,, for 60 min, 02t = 6.32 x lO’O, in a Ti-50 Beckman rotor). Studies with radioactively labelled Percoll (Klingeborn and Pertoft in “PercolP’; Methodology and Applica- tions” by Pharmacia) and electron microscopic analysis (Schumacher et al., 1978) showed that Percoll can be removed from samples without any residuals. The partic- ulate material was collected from the band above the hard Percoll pellet and sedimented by centrifugation at 15,000 x g,, for 30 min. These pellets were washed overnight with isoosmotic buffer, postosmicated, dehydrated, and flat-embedded in Medcast (Ted Pella, CA) resin. Sections from three different depths of the pellets were cut on a

Sorvall MT-I ultramicrotome and examined in a Siemens IA electron microscope after counterstaining with 10% uranyl acetate followed by Reynolds lead citrate (1963). Photographs of random fields (similar to those in Figs. 3 and 4) from three different groups of synaptosome preparations were made at magnifications of 7,300-54,000 di- ameters. A total of 43 photographs were randomly chosen for counting the synaptosomes. Assessment of samples was carried out essentially as has been previously described (Dodd et al., 1981). Synaptosomes were defined as membrane-bound, vesicle-containing structures.

RESULTS AND DISCUSSION

Isolation of synaptosomes Percoll, which has very low osmolality (<20

mOslkg HzO), can be made isoosmotic b y the addition of either physiological saline or tissue culture media. Since subcellular particles tend to aggregate in the presence of salts and because sucrose is as- sumed to have a stabilizing effect on synaptosomal membranes (Sperk and Baldessarini, 1977), 0.25 M sucrose was utilized in our experiments to adjust the Percoll solutions to isoosmicity. The preparation time from obtaining brain tissue to a usable synaptosomal fraction was approximately 1 h. We believe that this time reduction, in combination with maintaining the isoosmolarity of the gradient medium throughout the entire procedure, greatly increased the probability of preserving the intactness and the viability of the subcellular particles.

Our best subcellular resolution was achieved using swinging bucket rotors (Beckman SW 27.1 rotor). However, separation has also been accom- plished satisfactorily in fixed-angle rotors spun in a high-speed centrifuge (Sorvall SM-24 rotor). Accel- eration should be slow and no brake applied with both types of rotors ( 0 2 t = 1.59 x lo9 rad2/s).

In the isoosmotic Percollisucrose gradients at 4”C, synaptosomes sedimented near the interphase of the 10%/16% Percoll layers (Fig. 2). Calculated values for 10% and 16% Percoll in sucrose are 1.046 and 1.054 g/cm3; thus the synaptosomal density is within this range. This value (1.050 g/cm3) is sub- stantially lower than that determined for synaptosomes in sucrose gradients (1.13- 1.17 glcm3; Gurd et al., 1974), which indicates that the particles have not been dehydrated in the isotonic Percoll as they would be in the hyperosmotic sucrose (Whittaker, 1969; Breer et al., 1978). The low density of the particles also indicates that Percoll did not adhere to the synaptic membranes, as uncoated silica would (Lagercrantz and Pertoft, 1972).

Since Percoll is nontoxic to biological materials it was unnecessary to remove it from the purified preparations, except for electron microscopy.

Morphological and biochemical characterization of gradient fractions

When the crude mitochondria1 fraction (PJ of

1. Neurockem., Vol. 43, No. 4, 1984


brain tissue was separated on the Percoll isoosmotic step gradient three major bands appeared (Fig. 2). Morphologically (Fig. 3A), the band near the sur- face of the gradient, in the 7.5% layer (band A in Fig. 2 ) , consisted mainly of large membrane material. Synaptosomes were probably unsealed in this fraction, since we could detect only small amounts of occluded low-molecular-weight synaptosomal markers (5-HT; bound ATP and LDH; as shown in Fig. 2). The second band (B) was obtained from the 10% Percoll layer. Morphologically (Fig. 3B), band B showed a larger number of synaptosomes than band A, and it contained an increased amount of bound transmitter and ATP (Fig. 2). However, the contamination of this fraction with membrane fragments was considerable.

Band C, which was the most enriched in synaptosomes, occurred near the 10%/16% Percoll inter- face. As the distribution of the small-molecular- weight synaptosomal markers demonstrates (Fig.

0 1 0 :

1.0 j r--,

- _ 0.5 r r A 0.5

0 0 4.3 8.6 12 5

VOLUME [m i ]

P2 7 .5% 10% 16 % Percoll Conc.

FIG. 2. Distribution patterns of protein, LDH, bound ATP, and 5-HTafter centrifugation ondiscontinuous Percoll density gradients. The graph depicts a typical experiment. Tubes for Beckman rotor SW 27.1 were layered from the bottom with Percoll solutions of three concentrations: 4 rnl of 16%, 4 ml of 10%. and 4.5 ml of 7.5% (including 0.5 mi of P2). Fractions: (A), myelin and light membranes; (B), ’‘light’’ synaptosornes and membranes; (C), synaptosornes; (D). mitochondria. Composition of parent fraction (P,)/g of original tissue with percentage recoveries in parentheses: protein, 9.4 mg (95%); LDH activity, 6.5 pmol product formed/min/mg protein (101%); ATP, 3.8 nmol (81%); and 5-HT, 1960 relative fluorescence intensity (1 17%). Each assay was measured in du- plicate.

21, band C had the highest bound ATP and 5-HT contents. The distribution of two other transmitters (NA and dopamine) showed similar profiles (results not shown here). The occluded LDH activity was also the highest in this band. Morphologically, the pellet obtained after fixation of band C (Fig. 3C and Fig. 4) for electron microscopy was homogeneous through its depth and consisted primarily of synaptosomes (see also Table 1). Randomly cut sections through the entire synaptosomal pellet indicated that the primary contaminant consisted of nonsynaptic membrane-limited particles (probably microsomes). Small amounts of free mitochondria could also be observed. Myelin contamination was negligible. Noteworthy is that most of the synaptosomes were very densely packed with synaptic vesicles and contained well-preserved intrasynaptosomal mitochondria. Synaptosomes appeared intact and, because of the presence of 0.1 mM EDTA in all isolation media, adhering postsynaptic material was rarely seen (Van Leeuwen et al., 1976).

The pellet from the bottom of the step gradient (band D) consisted mainly of well-preserved extra- terminal mitochondria, but a number of larger synaptosomes also appeared here (Fig. 3D). The synaptosomal “contamination” of this fraction was considerably reduced for use as a reference fraction by increasing the concentration of the bottom Per- coll layer of the gradient (as discussed in Materials and Methods).

Quantitative evaluation by electron microscopy (Table 1) indicated that isolated nerve terminals made up at least 70% of the structures of fraction C. This estimated value falls in the middle range of earlier reported data. The purity of our fractions did not reach the values obtained by the continuous gradient technique (Whittaker, 1968), or by the Hajos (1975) method. It is noteworthy, however, that figures in the original Whittaker paper did not include the unidentified membrane particles, and a recent biochemical analysis of the Hajos preparation (Dodd et al., 1981) failed to prove the high purity results originally obtained by electron micros- COPY.

The composition of the remaining 30% of fraction C was similar to those reported for various other preparations. The free mitochondria1 contamination was 3%, and <3% of the total counted particles could be identified as myelin fragments in our synaptosomal fractions. The identity of the rest of the particles can be summarized as membranes of unidentified origin. Our fractions showed approximately 25% membrane contamination, further in- dicating that the quality of our preparations is comparable to that of previous works.

A selection of putative marker enzymes for various subcellular constituents such as mitochondria, microsomes, myelin, and glial and neural plasma membranes were assayed in the subfractions. Table

J. Neurochem.. Vol. 43, N o . 4 . 1984

I118 A . NAGY AND A . V . DELGADO-ESCUETA

FIG. 3. Electron micrographs of rat brain subcellular fractions isolated on a Percoll step gradient. The sections are from the middle of the pellet. Positions of fractions (A), (B), (C), and (D) are shown in Fig. 2. Fixation, staining, and embedding as described in Materials and Methods. x 17,000.

1. Neurochem., Vol. 43, No. 4, 1984

ISOLATION OF SYNAPTOSOMES ON PERCOLL GRADIENT 1119

FIG. 4. Typical fields of rat brain synaptosomal preparations isolated on a Percoll step gradient. A: Medium power ( x 34,000) general field of synaptosomal pellet. In this view all mitochondria are intrasynaptosomal. Quantitations were made from such sections. B: Low-magnification ( x 7,300) view of synaptosomal preparation. Several synaptosome profiles containing vesicles and mitochondria can be identified. Few free mitochondria and nonsynaptic-membrane-limited particles can also be seen. C: High-power ( x 54,000) view of synaptosome containing mitochondria and vesicles.

2 shows the marker enzyme activities that were measured in the synaptosomal preparations. The amount of contamination of synaptosomes by various nonsynaptosomal particles was calculated by taking the ratio of the specific activity of marker

enzymes measured in the synaptosomal fraction to that measured in the reference fraction. This value is expressed as a percentage.

The microsomal contamination of the synapto- soma1 fractions was assessed by measuring NCR

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1120 A. NAGY AND A . V . DELGADO-ESCUETA

TABLE 1. Morphological assessment of synaptosomal fractions

Distribution of subcellular structures as percentage of the total identified particles in synaptosomal preparations obtained by:

(1) ( 2 ) (3) Discontinuous Continuous Discontinuous Discontinuous PercolYsucrose sucrose sucrose Ficolllsucrose gradient (new) gradient gradient gradient

Particles (%) (%) (%) (%)

Synaptosomes 70.2 I 2.2 96-97" 48 60-70 Free mitochondria 3.3 i 0.6 1-3 4 10 Myelin 2.1 I 0.7 0-3 3 n.g. Unidentified 24.3 I 1.5 (1 45 20

Samples from band C of the gradients were taken as synaptosomes. Assessment of samples was carried out on photographs (total 43) of three different preparations as described in Materials and Methods. Values are the means I SEM expressed as percentage of the total assigned to the four groups of structures.

n.g., Not given. ( I ) Whittaker et al. (1968); "data in this column represent the proportion of identified particles only;

( 2 ) Dodd et al. (1981). (3) Cotman and Matthews (1971).

morphologically unidentified material was not included into these estimates of purity.

activity (Curd et al., 1974), and it amounted to approximately 4%. Although the present data are slightly lower than that reported by Booth and Clark (1978) (8%), we still have to conclude that microsomes are the major contaminants in our synaptosomal fractions. A 2.5% contamination was indicated for the mitochondrial outer membrane marker (rotenone-insensitive NADH:cytochrome c reductase). For the calculation of the specific activity of this enzyme in reference fractions a cor- rection was made based on the assumption that the outer mitochondrial membrane consists of 8% of the total mitochondrial protein (Brunner and Bucher, 1970). The specific enzyme activity found in our synaptosomes (8.3 nmolhidmg) was much lower than the value reported by Babitch et al. (1976) for chicken synaptosomal preparations (355 nmolhnidmg). However, when estimating the con-

tamination of free mitochondria, one has to con- sider that rotenone-insensitive NADH:cytochrome c reductase also occurs in microsomes (Beattie, 1968). Very low mitochondrial contamination was also indicated by assaying for two inner membrane markers, cytochrome c oxidase and SDH (results not shown here). The myelin marker, CNPase activity, was barely detectable in fraction C (12 nmol/ midmg). This result is in good agreement with the very few myelin-like fragments seen by electron microscopy. Corresponding values found by other authors are higher than ours (2.4-7.4 prnoYrninlmg; Morgan et al., 1971; Dodd et al., 1981). CNPase activity has also been found to be associated with neural and glial plasma membranes (Zanetta et al., 1972), and thus the actual myelin contamination of our synaptosomal preparation was probably even lower than the present estimate (Table 2). It is gen-

TABLE 2. Contamination of synaptosomal prepararions by other subcellular constituents

No. of Marker enzymes experiments Organelle

NCR 10 Microsomes NCR, rotenone-insensitive 4 Mitochondrion,

CNPase 6 Myelin and

Na+ ,K+-ATPase 5 Plasma

outer membrane

glia membrane

membrane

Specific marker enzyme activities in:

Percentage Intact contamination"

Organelle synaptosomes (%)

17.3 f 2.4 0.68 2 0.08 3.9

336.5 f 23.5 8.3 2 0.4 2.5

3,366.7' 12.0 % 2.7 0.3

1,344.7 I 296.7 10.6 I 2.8 0. I

The average specific enzyme activities (I SEM) are expressed in nmoYmin/mg of protein. The organelle (reference) fractions were

Percentage contamination is calculated as the ratio of specific activity of synaptosomal fraction to that of the reference fractions prepared as indicated in Materials and Methods.

(organelles). The total recoveries for the enzymes were in the range of 97410%. * Taken from Spohn and Davison (1972).

J . Neurochem., Vol. 43, N o . 4, 1984


erally accepted that ouabain-sensitive Na+ , K +-

ATPase is a marker for the neural plasma membrane, and that the active site of this enzyme is facing intrasynaptosomally. Therefore the low specific enzyme activity detected in fraction C (approximately 10 nmol/min/mg) indicates the presence of only a small number of broken synaptosomes and/or postsynaptic attachments. The disruption of synaptosomes resulted in a substantial (10- 15-fold) enhancement in the activity of Na+, K+-ATPase over that measured with intact synaptosomes. This later value was very similar to that found by Morgan et al. (1971) and Wood and Wyllie (1981) in their synaptosomal fractions.

In general, when marker enzyme activities of our synaptosomal preparations have been compared with those of other investigators, we found our values to be much lower. The differences may be partially explained by our maintaining isotonicity in all incubations by adding sucrose to the reaction mixtures.

Percoll is a polydisperse colloid, and when it is centrifuged at approximately 25,000 x g,, or higher (for Percoll in 0.25 M sucrose), its component particles will form a continuous gradient. Recent studies have utilized continuous Percoll gradients for the separation of rat brain subcellular structures from P, fractions (Shank and Campbell, 1983; Yu and Hertz, 1983); however, neither of these publi- cations include a biochemical characterization of the preparations, and thus we are unable to com- pare the purity of their subcellular particles to that of other preparations.

Integrity of synaptosomes Centrifugation in Percoll also resulted in selective

enrichment of intact synaptosomes in our preparations. It has been shown that injured cells and cell debris band at the top of Percoll gradients, in con- trast to their behaviour in gradients containing low- molecular-weight substances, such as sucrose (Per- toft et al., 1977). The cytosomal marker enzyme LDH was chosen as the biochemical indicator of synaptosome integrity (Johnson and Whittaker,

1963). Expression of the LDH activity of intact synaptosomes as a percentage of the total activity detected in disrupted synaptosomal fractions indicates the amount of plasma membrane leakage in the preparation (Marchbanks, 1967). As the data of Table 3 indicate, there was very little LDH activity present in the intact synaptosome preparations. Only about 2% of the total detectable synaptosomal LDH activity could be measured in intact preparations. Adam-Vizi and Marchbanks (1983) published similar observations regarding the amount of disrupted particles in their synaptosomal fractions (8%).

Synaptosomal fractions contain intrinsic endo- plasmic reticulum (Miller and Dawson, 1972) which can also be characterized by NCR activity. There- fore, unlike the NCR activity of reference micro- soma1 preparations, the NCR activity measured in synaptosomal preparations will be enhanced by the disruption of the particles. For this reason, we also attempted to measure the synaptosomal membrane integrity by monitoring the effect of plasma membrane disruption on NCR activity. The treatment of synaptosomes with Triton X-100 resulted in an approximately 12-fold increase in NCR activity. The actual changes in NCR activity caused by the disruption of synaptosomes is probably higher than that because Triton X-100, a detergent found to be suitable for mild disruption of subcellular particles, slightly inhibits NCR activity.

The condition of particles maintained in isoosmotic Percoll solution was very stable. LDH activity of intact particles did not exceed 5% of the total measurable activity 24 h after initial isolation. It is also noteworthy that high-speed pelleting of Percoll from fractions (for details see Materials and Methods) makes it possible to concentrate synaptosomes without packing them into a pellet, and thus, disruption that might occur as the result of other concentration procedures is avoided.

Synaptosomes prepared on isoosmotic Percoll gradients, similar to those separated on isoosmotic Ficoll gradients, and unlike those prepared on hyperosmotic sucrose gradients, are very sensitive

TABLE 3. Integrity of synaptosomes

in: Increase in Specific enzyme activities

enzyme activities Damaged Intact Disrupted by I% Triton-X-100 synaptosomes"

Enzymes synaptosomes synaptosomes (-fold) ( % r )

NCR 0.4 2 0.1 4.9 -c 0.2 12 8.2 LDH 12.5 f 1.8 539.1 f 50.3 49 2.3

Enzyme activity units are nmol/midmg of protein. Data represent the means ( f SEM) of enzyme activities determined from 10 (for LDH) or eight (for NCR) independent experiments. Total recoveries were 101% for LDH. and 1084 for NCR.

a Percentage damaged synaptosomes was calculated as the ratio of enzyme activities measured before and after disruption of particles with Triton X-100.

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I122 A . NAGY AND A . V. DELGADO-ESCUETA

TABLE 4. Yie ld of synaptosornal p repara t ions i so la ted on Percol l l sucrose gradients

No. of Yield Tissue source Type of tissue experiments (mgig wet weight tissue)

Rat Gray material of cerebral cortex 7

Hippocampus 4

cerebral cortex 3 Hippocampus 6

cortex 7

Mongolian gerbil Gray material of

Human Temporal

5.8 ? 0.3 5.2 -t 0.4

4.7 ? 0.6 2.1 -t 0.2

2.1 _f 0.2

Values are the means -C SEM

to osmotic shock (Jones, 1975). This is a favorable property when further subfractionation of synaptosomes is required.

Yield of preparations We have successfully applied the Percoll sepa-

ration technique to the isolation of synaptosomes from various mammalian brain tissues, including human brain specimens. The yield of synaptosomes obtained by this procedure was approximately 5 mg proteidg fresh rat brain cortex material (Table 4). The lower yield of gerbil hippocampal and human temporal lobe synaptosomes (approximately 2 mg/ g wet tissue) was probably the result of the presence of the large amount of white brain material in those tissue samples. Using rat brain, Dodd et al. (1981) and Wood and Wyllie (1981) obtained yields as high as 12 and 16 mglg wet weight tissue, while Babitch et al. (1976) produced 2.5 mg synaptosomes/g of chicken brain. The relatively low yield of synaptosomes from our preparations can be explained by increased purity (Morgan, 1976) and the loss of leaky synaptosomes.

The purity and the yield for synaptosomal preparations of small tissue samples (50.1 g), where the S, fraction was directly applied on Percoll gradients, were comparable to those shown in Tables 2 and 4.

The technique reported here offers a rapid, con- venient method of preparing highly purified synaptosomes from a large number of small tissue samples.

Acknowledgments: The authors would like to thank Dr. T. A. Shuster for helpful discussions and Ms. M. Akers for performing the electron microscopy. This project was funded at least in part with Federal grants from the Department of Health and Human Services under contract number NOl-NS-0-2332. The contents of this publication do not necessarily reflect the views or policies of the Department of Health and Human Ser- vices, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.

REFERENCES Abdel-Latif A. A. (1966) A simple method for isolation of nerve-

ending particles from rat brain. Biochim. Biophys. Actu 121, 403 - 406.

Adam-Vizi V. and Marchbanks R. M. (1983) Studies on the osmotic disruption and resealing of synaptosomes. J. Neuro- chem. 41, 780-785.

Autilio L. A,, Appel S. H., Pettis P., and Gambetti P. L. (1968) Biochemical studies of synaptosomes in v i t ro . I. Protein synthesis. Biochemistry 7, 2615-2622.

Babitch J. A., Breithaupt T. B., Tien-Cheng Chiu, Garadi R., and Helseth D. L. (1976) Preparations of chick brain synaptosomes and synaptosomal membranes. Biochim. Bio- phys. Acta 433, 75-89.

Beattie D. S. (1968) Enzyme localization in the inner and outer membranes of rat liver mitochondria. Biochem. Biophys. Res. Commun. 31, 90-907.

Booth R. F. G. and Clark J. B. (1978) A rapid method for the preparation of relatively pure metabolically competent synaptosomes from rat brain. Biochem. J. 176, 365-370.

Bradford M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal . B iochem. 72, 248-254.

Breer H., Morris S. J., and Whittaker V. P. (1978) A structural model of cholinergic synaptic vesicles from the electric organ of Torpedo marmoruta deduced from density mea- surements at different osmotic pressures. Eur. J. Biochem.

Brunner G. and Bucher Th. (1970) Determination of the quantitative relationship of outer and inner membrane proteins in rat liver mitochondria by means of enzymology and electron microscopy. FEBS Lett. 6, 125-131.

Chang C. C. (1964) A sensitive method for spectrofluorometric assay of catecholamines. I n t . J. Neuropharmucol. 3, 643-649.

Cooper A. J. and Perry S. (1980) The applicability of freeze-thaw percoll gradients to whole-cell isopycnic fractionations: preliminary results. J. Immunol. Merhods 37, 353-362.

Cooperstein S. J. and Lazaron A. (1951) A microspectrophoto- metric method for the determination of cytochrome oxidase. J . Biol. Chem. 189, 665-670.

Cotman C. W. and Matthews D. A. (1971) Synaptic plasma membranes from rat brain synaptosomes: isolation and partial characterization. Biochim. Biophys. Actu 249, 380-394.

Day E. D., McMillen P. N., Mickey D. D., and Appel S. H. (1971) Zonal centrifuge profiles of rat brain homogenates: instability in sucrose, stability in iso-osmotic Ficoll-sucrose. Anal. Bichem. 39, 29-45.

De Belleroche J. S. and Bradford H. F. (1973) The synaptosome: an isolated working, neuronal compartment, in Progress in Neurobio logy , Vol. 114. (Kerkut G. A. and Phillis J. W., eds), pp. 275-298. Pergamon, Oxford.

87, 453-458.

1. Neurochem., Vol. 43, No. 4, 1984


De Robertis E., Pellegrino de Iraldi A,, Rodriguez de Lorez Arnaiz G., and Salganicoff L. (1962) Cholinergic and non- cholinergic nerve endings in rat brain 1. Isolation and subcellular distribution of acetylcholine and acetylcholine es- terase. J. Neurochem. 9, 23-35.

Dodd, P. R., Hardy J. A., Oakley A. E., Edwardson J. A,, Perry E. K., and Delannoy J. P. (1981) A rapid method for preparing synaptosomes: comparison, with alternative procedures. Brain Res. 226, 107-118.

Dowdali M. J . , Boyne A. E, and Whittaker V. k? (1974) Aden- osine triphosphate: a constituent of cholinergic synaptic vesicles. Biochem. J. 140, 1-12.

Duncan H. M. and Machler B. (1966) Electron transport system of yeast. 111. Preparation and properties of cytochrome oxidase. J. Biol. Chem. 241, 1694-1697.

Earl D. C. N. and Korner A. (1965) The isolation and properties of cardiac ribosomes and polysomes. Biochem. J . 94, 721-734.

Gray E. G. and Whittaker V. P. (1962) The isolation of nerve endings from brain. An electron microscopic study of cell fragments divided by homogenization and centrifugation. J . Anur. (Lund.) 96, 79-88.

Gurd J . W., Jones L. R., Mahler H. R., and Moore W. J. (1974) Isolation and partial characterization of rat brain synaptic plasma membranes. J . Neurochem. 22, 281-290.

Hajos E (1975) An improved method for the preparation of synaptosomal fractions in high purity. Brain Res. 93, 485-489.

Hathaway P. W., Jakoi L., Troyer W. G., J r . , and Bogdonoff M. D. (1967) A method for semiautomatic differential analysis of urinary catecholamines. Anal . Biochem. 20, 466-476.

Hosie R. J. A. (1965) The localization of adenosine triphospha- tases in morphologically characterized subcellular fractions of guinea-pig brain. Biochem. J. 96, 404-412.

Johnson M. K. (1960) The intracellular distribution of glycolytic and other enzymes in rat brain homogenates and mitochondrial preparations. Biochem. J. 77, 610-618.

Johnson M. K . and Whittaker V. P. (1963) Lactate dehydrogenase as a cytoplasmic marker in brain. Biochem. J. 88, 404 -409.

Jones D. G. (1975) Synapses and synupfosomes: Morphological aspects (Jones D. G., ed), pp. 44-97. Chapman and Hall, London; Wiley, New York.

Kurokawa M. T., Sakamoto M. T., and Kato M. (1965) Distri- bution of sodium-plus-potassium-stimulated adenosine-tri- phosphatase activity in isolated nerve-ending particles. Biochem. J . 97, 833-844.

Lagercrantz H. and Pertoft H. (1972) Separation of catechol- amine storing synaptosomes in colloidical silica density gradients. J . Neurochem. 19, 81 1-823.

Lanzetta P. A,. Alvarez L. J., Reinach P. S.. and Candia 0. A. (1979) An improved assay for nanomole amounts of inor- ganic phosphate. Anal. Biochem. 100, 95-97.

Marchbanks R. M. (1967) The osmotically sensitive potassium and sodium compartments of synaptosomes. Biochem J . 104, 148-157.

Mateyko G. M. and Kopac M. J. (1963) Cytophysical studies on living normal and neoplastic cells. Ann. NYAcad . Sci. 105, 185-2 18.

Miller E. K. and Dawson R. M. C. (1972) Can mitochondria and synaptosomes of guinea pig brain synthesize phospholipids? Biochem. J . 126, 805-821.

Morgan 1. G. (1976) Synaptosomes and cell preparation. Com- mentary. Neuroscience 1, 159- 165.

Morgan I . G., Wolfe L. S. , Mandel P., and Gombos G. (1971) Isolation of plasma membranes from rat brain. Biochim. Biophys. A c f a 241, 737-751.

Nagy A. and Delgado-Escueta A. V. (1983) Rapid preparation of synaptosomes from mammalian brain. Ninth Meeting of the

International Society for Neurochemistry. (Abstr) J . Neu- rochem. 41, (Suppl), S55.

Olafson R. W., Drummond G. I . , and Lee J. F. (1969) Studies on 2' , 3'-cycIic nucleotide-3'-phosphohydrolase from brain. Can. 1. Biochem. 47, 961-966.

Omura T. and Takesue S. (1970) A new method for simultaneous purification of cytochrome 6 , and NADPH-cytochrome c reductase from rat liver microsomes. J . Biochem. (Tokyo) 61, 249-257.

Pertoft H. , and Laurent T. C. (1982) Sedimentation of cells in colloidal silica (Percoll), in Cell Separation: Mefhods and Selected Applicafions, Vol. I . (Pretlow T. G . and Pretlow T. P., eds) Academic Press, New York.

Pertoft H., Rubin K., Kjellen L., Laurent T. C., and Klingeborn B. (1977) The viability of cells grown or centrifuged in a new density gradient medium, PercolP. Exp. Cell. Res . 110, 449-457.

Peterson G. L . (1977) A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal. Biochem. 83, 346-356.

Reynolds E. S. (1963) The use of lead citrate at high pH as an electro-opaque stain in electron microscopy. J . Cell Biol. 17, 208-212.

Schumacher M., Schafer G., Holstein A. F., and Hilz H. (1978) Rapid isolation of mouse Leydig cells by centrifugation in Percoll density gradients with complete retention of morphological and biochemical integrity. FEBS L e u . 91, 333-338.

Shank R. A. and Campbell G. Le M. (1983) Ornithine as a pre- cursor of glutamate and GABA: uptake and metabolism by neuronal and glial enriched cellular material. J . Neurosci. Res. 9, 47-57.

Snyder S. H., Axelrod J., and Zweig M. (1965) A sensitive and specific fluorescence assay for tissue serotonin. Biochem. Pharmacol. 14, 831-835.

Sperk G. and Baldessarini R. J. (1977) Stabilizing effect of sucrose on leakage of cytoplasm from rat brain synaptosomes in saline media. J . Neurochem. 28, 1403-1405.

Spohn M. and Davison A. N. (1972) Separation of myelin fragments from the central nervous system, in Research Mefhods in Neurochemisfry, Vol. I . , pp. 33-43. (Marks N., andRodnight R., eds), pp. 33-43. Plenum Press, New York.

Stanley P. E. and Williams S. G. (1969) Use of liquid scintillation spectrometer for determining adenosine triphosphate by the luciferase enzyme. Anal. Biochem. 29, 381-392.

Van Leeuwen C., Stam H., and Destreicher A. B. (1976) Isola- tion and partial characterization of chick brain synaptic plasma membranes. Biochim. Biophys. Acfu 436, 53-67.

Verity M. A. (1972) Cation modulation of synaptosomal respi- ration. J . Neurochem. 19, 1305-1317.

Whittaker V. P. (1968) The morphology of fractions of rat fore- brain synaptosomes separated on continuous sucrose density gradients. Biochem. J . 106, 412-417.

Whittaker V. P. (1969) The synaptosome, in Handbook of Neu- rochemisfry (Lajtha A,, ed), pp. 327-364. Plenum Press, New York.

Wolff D. A. (1975) The separation of cells and subcellular particles by colloidal-silica density centrifugation, in Mefhods in Cell Biology. Vol. I0 (Prescott D. M., ed). Academic Press, New York.

Wood M. D. and Wyllie M. G. (1981) The rapid preparation of synaptosomes, using a vertical rotor. J Neurochem. 37, 795-797.

Yu P. H. and Hertz L. (1983) Studies of the type A and type B monoamine oxidase. A new approach to their development and subcellular localization. Mod. Prohl. Phurmacopsy- chiafry 19, 177-188.

Zanetta J. P., Benda P., Gombos G.. and Morgan 1. G. (1972) The presence of 2'. 3'-cyclic AMP 3'-phosphohydrolase in glial cells in tissue culture. J . Nertroc-hem. 19, 881-883.

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Rapid Preparation of Synaptosomes from Mammalian Brain Using Nontoxic Isoosmotic Gradient Material (Percoll)