Neurochemical Research (1) 83-92 (1976)
C O M P A R A T I V E S T U D I E S ON S Y N A P T O S O M E S : A P P L I C A B I L I T Y OF THE R A P I D M E T H O D FOR P R E P A R I N G S Y N A P T O S O M E S TO E L A S M O B R A N C H BRAIN E.J.
SIMON, V . P . WHITTAKER1, H . MEILMAN, H. SHER~ G A l L V I C K E R S , a n d E . COUCH Marine Biological Laboratory Woods Hole, Massachusetts 02543
Accepted December 22, 1975
A flotation technique for the rapid preparation of synaptosomes, originally developed for invertebrate nervous tissue, has now been successfully applied to that of an elasmobranch fish (Mustelis canis, dogfish). The technique involves submitting the supernatant, obtained after a homogenate has been centrifuged at low speed to remove nuclei and tissue debris to centrifugal fields of intermediate intensity (106 g/rain), appears to separate well-sealed synaptosomes from those less well sealed as judged by the criteria of osmotic shrinkage, enzyme occlusion, and choline uptake. The sealed synaptosomes do not equilibrate with the 0.8 M sucrose used as the homogenization medium and rise to form a coherent peUicle at the top of the tube. Due to the short (1-1~ hr) preparation time, such synaptosomes may well prove useful in further metabolic studies.
INTRODUCTION Recent work with nervous tissue from squid (1) and lobster (2) has shown that synaptosomes can be isolated rapidly from such tissues by a flotation technique in which homogenates in 0.7-0.8 M sucrose (media 1 Requests for reprints should be addressed to Dr. V.P. Whittaker, Abteilung Neurochemie, Max-Planck-Institut for biophysicalische Chemie, Postfach 968, D-3400 Gottingen, Federal Republic of Germany.
83 (~ 1976PlenumPublishingCorporation,227 West 17thStreet, New York, N.Y. 10011. No part of this publicationmay be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic,mechanical, photocopying,microfilming,recording,or otherwise,withoutwrittenpermissionof the publisher.
84
E. J. S I M O N ET AL.
almost isoosmotic to the body fluids of these invertebrates) are centrifuged at low speed to remove nuclei and cell debris and then at 10,000-17,000g for 30-60 rain, whereupon the synaptosomes rise to the top of the centrifuge tube to form a firm peUicle which is readily collected for resuspension and metabolic studies. Since such preparations from the squid show a high, stable respiration when warmed in artificial sea water and are metabolically very active in bringing about the carrier-mediated uptake of choline (3), noradrenaline (4), and the incorporation of radioactive amino acids into protein via a Na-dependent, RNase insensitive, cycloheximide sensitive pathway (5), it occurred to us to investigate whether a similar flotation method could be applied to vertebrate nervous tissue. Since preliminary results (G. Vickers, unpublished) with guinea pig cerebral cortical tissue proved disappointing, we turned our attention to an elasmobranch (dogfish, Mustelis canis) which, unlike most vertebrates, has body fluids with an osmotic pressure of over 800 mOsmol/liter, and thus more closely resembles in this respect the invertebrate species to which the technique has so thr been successfully applied. We selected the olfactory lobes of the dogfish because preliminary work (E.J. Simon, H. Sher, and H. Meilman, unpublished) has indicated these as a relatively rich source of a neural component that stereospecifically binds morphine and related opiates, but the identification of the cellular localization of this component was being impeded by a lack of knowledge of the effect of homogenization on the tissue and the types and properties of subcellular particles generated. Published work on fish brain has so far been confined to teleosts (7). Our results indicate that well-sealed synaptosomes can indeed be obtained from dogfish olfactory lobes in reasonable yield and purity in a single step in just over 1 hr. Such synaptosome preparations from a vertebrate species may well have advantages for metabolic studies over conventional mammalian preparations requiring 2 hr or more. The study of the subcellular distribution of the opiate receptor in dogfish olfactory lobes will be reported separately.
EXPERIMENTAL PROCEDURE
Dissection, Homogenization, and Fractionation Freshly caught dogfish (Mustelis canis) were decapitated; the olfactory lobes (wet wt. 1.8--2.2 g) removed as rapidly as possible, chilled on ice, weighed, and homogenized in ice-
ELASMOBRANCH SYNAPTOSOMES
85
cold 0.8 M sucrose in an Aldridge homogenizer (all-round clearance, 1%; speed approximately 900 rpm, 12 up-and-down strokes) to give an approximately 10% w/v homogenate. After removing a sample (5 ml) for analysis, the homogenate was fractionated at (g4~ essentially as previously described for squid optic ganglia (1). It was first centrifuged at 1000g for 10 min to remove nuclei and cell debris: the resultant pellet was usually washed once by resuspending it in 0.8 M sucrose and recentrifuging; the washings were added to the original supernatant and the pellet was resuspended in a small (5-6 ml) volume for analysis (fraction P1). The combined supernatants were then centrifuged at 17,000g for 1 hr, whereupon a peUicle of floating material formed at the top of the tube and a pellet formed at the bottom. These were separated from the relatively clear fluid between (fraction S~), rinsed with 0.8 M sucrose (the rinsings were discarded), and suspended in small (2-3 ml) volumes of 0.8 M sucrose to give fractions P2L and P~H, respectively. When fraction P2L was required for metabolic studies the procedure was abbreviated by centrifuging the homogenate immediately at 17,000g for 1 hr to give a combined PI - P2H, a P~L, and an $2 fraction.
Marker Enzyme, Acetylcholine, and Protein Determinations Lactate dehydrogenase, a marker for soluble cytoplasm, was measured spectrophotometrically by following the transfer, in 0.8 M sucrose, of hydrogen from N A D H + to pyr~Jvate at 340 nm. The enzyme was measured in the presence of 1% Triton X-100 to release any occluded enzyme. The difference in enzyme activity in the presence and absence of detergent gave a measure of occluded lactate dehydrogenase. Malate hydrolyase (fumarase), a mitochondrial marker, was also measured spectroscopically by following the formation of the double bond of fumarate at 250 nm in the presence of 1% Triton X-100. The dogfish enzyme was found to be rather unstable, which led to low recoveries. Acetylcholine, a synaptosomal marker, was assayed on a thin strip of the dorsal muscle of the leech, after release from fractions by heating them at pH 4 and 100~ for 10 min. Difficulty was experienced in performing accurate assays on samples containing high concentrations of sucrose, particularly when (as in fraction $2) the concentration of acetylcholine was low. This accounts for the large variance in the recoveries; nevertheless, the distribution of acetylcholine among the various fractions was reasonably consistent from experiment to experiment (Table I). Total tissue acetylcholine was determined by means of a standard trichloracetic acid extraction procedure (8). Protein was estimated by the Lowry method. Full details and references have been given elsewhere. (9).
Uptake of Choline Choline uptake into synaptosome fractions was measured at 24~ essentially previously (3) described using [N-Me-3H] choline (0.1 /~Ci at a final concentration of ~M). Sucrose suspensions were diluted 1:10 with Tris-buffered artificial sea-water elasmobranch Ringer solution to give a final concentration of fraction (P2L or
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equivalent to 10-20 mg of tissue. Controls were diluted with distilled water. Uptake was measured in 1 ml of suspension and terminated by filtration through MiUipore filters (0.45p.m pore diameter). For counting, the filters after washing with suspension medium (3• 5 rot), were dissolved in 1.0 ml of methylcellosolve before adding 10 ml of Triton-toluene scintillation fluid. Counting efficiency was 40% as determined by the external standard ratio.
Preparation of Samples for Electron Microscopy Small pieces of the floating pellicle from which fraction P2Lwas derived by suspension in 0.8 M sucrose were floated into ice-cold 2% glutaraldehyde in 0.7 M sucrose--0.]l M S0rensen's phosphate buffer, pH 7.2, containing approximately 0.3 mM CaCI2 (SPC). After 1 hr the fragments of pellicle were washed four times for 15 rain in SPC, postfixed (1 hr) in a% OsO4 in SPC (10), dehydrated in propylene glycol, embedded in Araldite 502, and stained with lead citrate (11). Thin sections were examined in a Philips-300 electron microscope at 60-80 kV. Fraction $2 was prepared slightly differently: it was mixed with 4 vol 3.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2, containing 0.3 M sucrose-10 mM CaCI2. After 3 hr at 0~ particulate material was sedimented (50,000g for 60 rain), the supernatant poured off, and the pellet washed three times for 45 rain with 0.2 M sodium cacodylate buffer, pH 7.2, containing 0.5 M sucrose and 10 mM CaC12, then postfixed (4hr) in 1% OsO4 in 0.I M cacodylate buffer, pH 7.2, containing 0.4 M sucrose and 5 mM CaC12, washed with 0.1 M sodium acetate-10 mM CaC12, stained in 0.5% uranyl acetate in 0.05 M sodium acetate,--5 mM CaC12 (3 hr), washed with 0.1 M sodium acetate, set in 0.5% Agar, dehydrated, and embedded.
RESULTS
AND
DISCUSSION
Table I summarizes the distribution of lactate dehydrogenase, fumara s e , a c e t y l c h o l i n e , a n d p r o t e i n in t h e v a r i o u s f r a c t i o n s . I t will b e s e e n t h a t as w i t h t h e f r a c t i o n s f r o m s q u i d o p t i c g a n g l i a (1) a n d l o b s t e r v e n t r a l n e r v e c o r d (2), t h e m a t e r i a l c o l l e c t e d as a p e l l i c l e f l o a t i n g o n t h e 0.8 M s u p e r n a t a n t ( f r a c t i o n P2L) is rich in a c e t y l c h o l i n e . E l e c t r o n m i c r o g r a p h s o f this f r a c t i o n ( F i g u r e l) s h o w e d t h a t it is r e l a t i v e l y rich in s y n a p t o s o m e s , w i t h r e l a t i v e l y little c o n t a m i n a t i o n b y m y e l i n o r m i c r o s o m a l m a t e r i a l . T h e s y n a p t o s o m e s s o m e t i m e s h a v e w h a t a p p e a r to b e fragm e n t s o f d e n d r i t e s o r d e n d r i t e s p i n e s a d h e r i n g to t h e m , a n d o t h e r profiles may represent dendritic fragments free of synaptic attachments in t h e p l a n e o f s e c t i o n . T h i s f r a c t i o n r e p r e s e n t s o n l y a b o u t 23% o f t h e t o t a l r e c o v e r e d p r o t e i n , y e t c o n t a i n s , o n t h e a v e r a g e , 43% o f the recovered acetylcholine. Thus the relative specific concentration of a c e t y l c h o l i n e in t h e f r a c t i o n is w e l l a b o v e u n i t y , i n d i c a t i n g a c o n s i d e r a b l e e n r i c h m e n t o f c h o l i n e r g i c t e r m i n a l s in this f r a c t i o n . T h e a c e t y l c h o line c o n t e n t o f t h e h o m o g e n a t e s w a s n o t s i g n i f i c a n t l y d i f f e r e n t f r o m t h a t o f w h o l e t i s s u e ( T a b l e I), s h o w i n g t h a t t h e l a t t e r d o e s n o t c o n t a i n f r e e
88
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acetylcholine. This indicates that as in mammalian caudate nucleus (12) cholinergic neurons are represented mainly by their terminals and that synaptosome formation has occurred in high yield with little loss of transmitter (for discussion see ref. 13). Lactate dehydrogenase, as would be expected of a soluble cytoplasmic marker, is mainly recovered in fraction $2; however, a significant proportion occurs in fraction P2L where it no doubt represents soluble cytoplasm sequestered within synaptosomes. Significantly, this fraction of lactate dehydrogenase is largely occluded (Table I) in contrast to that in the supernatant (Sz) fraction, which is almost entirely free. Somewhat puzzling, initially, was the considerable amount of acetylcholine found in fraction $2. Since the fraction contains acetylcholinesterase [the activity using acetylthiocholine as substrate (14) was 0.8 /~mol/mg of protein/min, about half that of mammalian cortex] and no anticholinesterase was added, this acetylcholine cannot be free: electron microscopy of the pellet (Figure 2) obtained when this fraction was fixed with glutaraldehyde and sedimented at 50,000g for 1 hr revealed the presence of synaptosomes heavily contaminated with membrane fragments. The external membranes of these synaptosomes often showed evidence of blebbing or discontinuities. We therefore conclude that the acetylcholine of this fraction is associated with incompletely sealed or partially disrupted synaptosomes. Such structures would not show latency or occlusion with respect to their content of lactate dehydrogenase or other soluble enzymes and would rapidly equilibrate with 0.8 M sucrose, thus losing their bouyancy, the property necessary for flotation and pellicle formation. Difficulties were encountered in measuring fumarase levels which were low in all fractions under the assay conditions used. The distribu-
T A B L E II UPTAKE OF CHOLINE BY DOGFISH OLFACTORY LOBE SYNAPTOSOMES a U p t a k e o f choline (pmol/min/g tissue) at 24~ in Suspension medium Tris-buffered s e a water E l a s m o b r a n c h Ringer
Fraction P2L 16.4 --
Fraction P2L-fast b 8.1 13.6
Fraction S2-fast ~ -0
Rates o f uptake refer to a 10-min incubation period. b In t h e s e e x p e r i m e n t s , the h o m o g e n a t e was centrifuged immediately at 17,000g for 1 hr to form a combined P1 + P~H pellet.
90
E. J. S I M O N E T AL.
=o_
ELASMOBRANCH SYNAPTOSOMES
91
tion indicates that fraction PzH, as in the squid, is enriched in mitochondria. However, electron micrography, while confirming this, showed the presence of not inconsiderable numbers of synaptosomal profiles and membrane fragments some of which were apparently of synaptosomal origin. Choline Uptake. Uptake of choline by fraction P~L was linear for the first 10 rain and thereafter showed some fall in rate. Initial rates are shown in Table II. These are comparable to those observed with mammalian synaptosome preparations of similar acetylcholine content (e.g., rabbit cerebellum 24 pmol/min/g at 25~ M.J. Dowdall, personal communication). Osmotic shock caused 90-100% inhibition and hemicholinium (100/zM), 50-90% inhibition. Noteworthy was the complete lack of uptake by fraction Sz, confirming the impression that the synaptosomes in this fraction are incompletely sealed. A kinetic analysis of the uptake of choline by fraction P2L will be required to determine whether this fraction contains the high-affinity uptake system specific for cholinergic synaptosomes (3, 15, 16), as well as the more widely distributed low-affinity system. However, at the choline concentration used (1.0 /zM), uptake would occur equally via both systems if dogfish synaptosomes are similar to others examined. A preparation of synaptosomes from a vertebrate obtained in a single step by a relatively rapid procedure which apparently selects well-sealed structures may have advantages in the study of metabolic processes, especially those dependent on labile components.
ACKNOWLEDGMENTS We thank Joe Keeter, Deparment of Neuroscience, Albert Einstein College of Medicine, for electron microscopy of fraction $2. This work was supported by grant No. DA 00017 from the National Institute on Drug Abuse. E.J.S. is a career scientist of the Health Research Council of the City of New York.
REFERENCES 1. DOWDALL,M.J., and WHITTAKER,V.P. 1973. Comparative studies on synaptosome formation: The preparation of synaptosomes from the head ganglion of the squid,
Loligo pealii. J. Neurochem. 20:921-935. 2. NEWKIRK, R.F., BALLOU, E.W., VICKERS, G., and WHITTAKER, V.P. 1976. Comparative studies in synaptosome formation: Preparation of synaptosomes from the ventral nerve cord of the lobster (Homarus americanus). Brain Res. 101:103-111.
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E. J. SIMON ET AL.
3. DOWDALL, M.J., and SIMON, E.J. 1973. Comparative studies on synaptosomes: 4.
5.
6.
7. 8. 9.
10. 11. 12.
13. 14.
15.
16.
Uptake of [N-Me-3H]choline by synaptosomes from squid optic lobes. J. Neurochem. 21:969-982. POLLARD, H.B., BOHR, V.A., DOWDALL, M.J., and WHITTAKER, V.P- 1973. A chlorpromazine-sensitive, high affinity uptake system for L-noradrenaline in synaptosomes from the optic lobe of squid. Biol. Bull. (Woods Hole) 145:449-450. HERN/~NDEZ, A.G., LANGFORD, G.M., MARTINEZ, J.L., and DOWDAEL, M.J. 1976. Protein synthesis by synaptosomes from the head ganglion of the squid, Lotigo pealii (submitted for publication). SIMON, E.J., HILLER, J.M., and EDELMAN, I. 1973. Solubilizafion of a stereospecific [aH]etomorphine-macromolecular complex from a rat brain membrane fraction. Proc. Natl. Acad. Sci., U.S.A. 70:1947-1949. WHITTAKER, V.P., and GREENGARD,P. 1971. The isolation of synaptosomes from the brain of a teleost fish, (Centriopristes striatus). J. Neurochem. 18:173-176. MACINTOSH, F.C., and PERRY, W.L.M. 1961. Biological estimation of acety!choline. Methods Med. Res. 3:78-92. WHITTAKER, V.P., and BARKER, L.A. 1972. The subcellular fractionation of brain tissue with special reference to the preparation of synaptosomes and their component organelles. Pages 1-52, in FRIED, R., (ed.), Methods in Neurochemistry, Vol. 2, Marcel Dekker, New York. MILLONIG, G . 1961. Advantages of a phosphate buffer for osmium tetroxide solutions in fixation. J. Appl. Phys. 32: 1637. REYNOLDS, E.S. 1963. The use of lead citrate at high pH as an electron-opaque stair in electron microscopy. J. Cell Biol. 17:208-212. LAVERTY,R., MICHAELSON,I.A., SHARMAN,D. F., and WHITTAKER, V.P. 1963. The subcellular localization of dopamine and acetylcholine in the dog candate nucleus. Br. J. Pharmacol. 21:482-490. WHITTAKER, V.P. 1971. Subcellular localization of neurotransmitters, Adv. Cytopharmacol. 1:319-330. ELLMAN, G.L., COURTNEY, K.D., ANDRES, V., and FEATHERSTONE,R.M. 1961. A new and rapid colorimetric determination of acetylcholinesterase activity, Biochem. Pharmacol. 7:88-95. DOWDALL, M.J., WACHTEER, K., and HENDERSON, F. 1975. Comparative studies on the high affinity choline uptake system. Abstr. 5th Int. Meet. Int. Soc. Neurochem., Barcelona, p. 122. DOWDALL, M.J. 1976. Synthesis and storage of acetylcholine in cholinergic nerve terminals. Pages 585-607, in BERL, S., CLARKE, D.D., and SCHNEIDER, O. (eds.), Metabolic Compartmentation in the Brain, Plenum Press, New York.
C O M P A R A T I V E S T U D I E S ON S Y N A P T O S O M E S : A P P L I C A B I L I T Y OF THE R A P I D M E T H O D FOR P R E P A R I N G S Y N A P T O S O M E S TO E L A S M O B R A N C H BRAIN E.J.
SIMON, V . P . WHITTAKER1, H . MEILMAN, H. SHER~ G A l L V I C K E R S , a n d E . COUCH Marine Biological Laboratory Woods Hole, Massachusetts 02543
Accepted December 22, 1975
A flotation technique for the rapid preparation of synaptosomes, originally developed for invertebrate nervous tissue, has now been successfully applied to that of an elasmobranch fish (Mustelis canis, dogfish). The technique involves submitting the supernatant, obtained after a homogenate has been centrifuged at low speed to remove nuclei and tissue debris to centrifugal fields of intermediate intensity (106 g/rain), appears to separate well-sealed synaptosomes from those less well sealed as judged by the criteria of osmotic shrinkage, enzyme occlusion, and choline uptake. The sealed synaptosomes do not equilibrate with the 0.8 M sucrose used as the homogenization medium and rise to form a coherent peUicle at the top of the tube. Due to the short (1-1~ hr) preparation time, such synaptosomes may well prove useful in further metabolic studies.
INTRODUCTION Recent work with nervous tissue from squid (1) and lobster (2) has shown that synaptosomes can be isolated rapidly from such tissues by a flotation technique in which homogenates in 0.7-0.8 M sucrose (media 1 Requests for reprints should be addressed to Dr. V.P. Whittaker, Abteilung Neurochemie, Max-Planck-Institut for biophysicalische Chemie, Postfach 968, D-3400 Gottingen, Federal Republic of Germany.
83 (~ 1976PlenumPublishingCorporation,227 West 17thStreet, New York, N.Y. 10011. No part of this publicationmay be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic,mechanical, photocopying,microfilming,recording,or otherwise,withoutwrittenpermissionof the publisher.
84
E. J. S I M O N ET AL.
almost isoosmotic to the body fluids of these invertebrates) are centrifuged at low speed to remove nuclei and cell debris and then at 10,000-17,000g for 30-60 rain, whereupon the synaptosomes rise to the top of the centrifuge tube to form a firm peUicle which is readily collected for resuspension and metabolic studies. Since such preparations from the squid show a high, stable respiration when warmed in artificial sea water and are metabolically very active in bringing about the carrier-mediated uptake of choline (3), noradrenaline (4), and the incorporation of radioactive amino acids into protein via a Na-dependent, RNase insensitive, cycloheximide sensitive pathway (5), it occurred to us to investigate whether a similar flotation method could be applied to vertebrate nervous tissue. Since preliminary results (G. Vickers, unpublished) with guinea pig cerebral cortical tissue proved disappointing, we turned our attention to an elasmobranch (dogfish, Mustelis canis) which, unlike most vertebrates, has body fluids with an osmotic pressure of over 800 mOsmol/liter, and thus more closely resembles in this respect the invertebrate species to which the technique has so thr been successfully applied. We selected the olfactory lobes of the dogfish because preliminary work (E.J. Simon, H. Sher, and H. Meilman, unpublished) has indicated these as a relatively rich source of a neural component that stereospecifically binds morphine and related opiates, but the identification of the cellular localization of this component was being impeded by a lack of knowledge of the effect of homogenization on the tissue and the types and properties of subcellular particles generated. Published work on fish brain has so far been confined to teleosts (7). Our results indicate that well-sealed synaptosomes can indeed be obtained from dogfish olfactory lobes in reasonable yield and purity in a single step in just over 1 hr. Such synaptosome preparations from a vertebrate species may well have advantages for metabolic studies over conventional mammalian preparations requiring 2 hr or more. The study of the subcellular distribution of the opiate receptor in dogfish olfactory lobes will be reported separately.
EXPERIMENTAL PROCEDURE
Dissection, Homogenization, and Fractionation Freshly caught dogfish (Mustelis canis) were decapitated; the olfactory lobes (wet wt. 1.8--2.2 g) removed as rapidly as possible, chilled on ice, weighed, and homogenized in ice-
ELASMOBRANCH SYNAPTOSOMES
85
cold 0.8 M sucrose in an Aldridge homogenizer (all-round clearance, 1%; speed approximately 900 rpm, 12 up-and-down strokes) to give an approximately 10% w/v homogenate. After removing a sample (5 ml) for analysis, the homogenate was fractionated at (g4~ essentially as previously described for squid optic ganglia (1). It was first centrifuged at 1000g for 10 min to remove nuclei and cell debris: the resultant pellet was usually washed once by resuspending it in 0.8 M sucrose and recentrifuging; the washings were added to the original supernatant and the pellet was resuspended in a small (5-6 ml) volume for analysis (fraction P1). The combined supernatants were then centrifuged at 17,000g for 1 hr, whereupon a peUicle of floating material formed at the top of the tube and a pellet formed at the bottom. These were separated from the relatively clear fluid between (fraction S~), rinsed with 0.8 M sucrose (the rinsings were discarded), and suspended in small (2-3 ml) volumes of 0.8 M sucrose to give fractions P2L and P~H, respectively. When fraction P2L was required for metabolic studies the procedure was abbreviated by centrifuging the homogenate immediately at 17,000g for 1 hr to give a combined PI - P2H, a P~L, and an $2 fraction.
Marker Enzyme, Acetylcholine, and Protein Determinations Lactate dehydrogenase, a marker for soluble cytoplasm, was measured spectrophotometrically by following the transfer, in 0.8 M sucrose, of hydrogen from N A D H + to pyr~Jvate at 340 nm. The enzyme was measured in the presence of 1% Triton X-100 to release any occluded enzyme. The difference in enzyme activity in the presence and absence of detergent gave a measure of occluded lactate dehydrogenase. Malate hydrolyase (fumarase), a mitochondrial marker, was also measured spectroscopically by following the formation of the double bond of fumarate at 250 nm in the presence of 1% Triton X-100. The dogfish enzyme was found to be rather unstable, which led to low recoveries. Acetylcholine, a synaptosomal marker, was assayed on a thin strip of the dorsal muscle of the leech, after release from fractions by heating them at pH 4 and 100~ for 10 min. Difficulty was experienced in performing accurate assays on samples containing high concentrations of sucrose, particularly when (as in fraction $2) the concentration of acetylcholine was low. This accounts for the large variance in the recoveries; nevertheless, the distribution of acetylcholine among the various fractions was reasonably consistent from experiment to experiment (Table I). Total tissue acetylcholine was determined by means of a standard trichloracetic acid extraction procedure (8). Protein was estimated by the Lowry method. Full details and references have been given elsewhere. (9).
Uptake of Choline Choline uptake into synaptosome fractions was measured at 24~ essentially previously (3) described using [N-Me-3H] choline (0.1 /~Ci at a final concentration of ~M). Sucrose suspensions were diluted 1:10 with Tris-buffered artificial sea-water elasmobranch Ringer solution to give a final concentration of fraction (P2L or
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87
equivalent to 10-20 mg of tissue. Controls were diluted with distilled water. Uptake was measured in 1 ml of suspension and terminated by filtration through MiUipore filters (0.45p.m pore diameter). For counting, the filters after washing with suspension medium (3• 5 rot), were dissolved in 1.0 ml of methylcellosolve before adding 10 ml of Triton-toluene scintillation fluid. Counting efficiency was 40% as determined by the external standard ratio.
Preparation of Samples for Electron Microscopy Small pieces of the floating pellicle from which fraction P2Lwas derived by suspension in 0.8 M sucrose were floated into ice-cold 2% glutaraldehyde in 0.7 M sucrose--0.]l M S0rensen's phosphate buffer, pH 7.2, containing approximately 0.3 mM CaCI2 (SPC). After 1 hr the fragments of pellicle were washed four times for 15 rain in SPC, postfixed (1 hr) in a% OsO4 in SPC (10), dehydrated in propylene glycol, embedded in Araldite 502, and stained with lead citrate (11). Thin sections were examined in a Philips-300 electron microscope at 60-80 kV. Fraction $2 was prepared slightly differently: it was mixed with 4 vol 3.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2, containing 0.3 M sucrose-10 mM CaCI2. After 3 hr at 0~ particulate material was sedimented (50,000g for 60 rain), the supernatant poured off, and the pellet washed three times for 45 rain with 0.2 M sodium cacodylate buffer, pH 7.2, containing 0.5 M sucrose and 10 mM CaC12, then postfixed (4hr) in 1% OsO4 in 0.I M cacodylate buffer, pH 7.2, containing 0.4 M sucrose and 5 mM CaC12, washed with 0.1 M sodium acetate-10 mM CaC12, stained in 0.5% uranyl acetate in 0.05 M sodium acetate,--5 mM CaC12 (3 hr), washed with 0.1 M sodium acetate, set in 0.5% Agar, dehydrated, and embedded.
RESULTS
AND
DISCUSSION
Table I summarizes the distribution of lactate dehydrogenase, fumara s e , a c e t y l c h o l i n e , a n d p r o t e i n in t h e v a r i o u s f r a c t i o n s . I t will b e s e e n t h a t as w i t h t h e f r a c t i o n s f r o m s q u i d o p t i c g a n g l i a (1) a n d l o b s t e r v e n t r a l n e r v e c o r d (2), t h e m a t e r i a l c o l l e c t e d as a p e l l i c l e f l o a t i n g o n t h e 0.8 M s u p e r n a t a n t ( f r a c t i o n P2L) is rich in a c e t y l c h o l i n e . E l e c t r o n m i c r o g r a p h s o f this f r a c t i o n ( F i g u r e l) s h o w e d t h a t it is r e l a t i v e l y rich in s y n a p t o s o m e s , w i t h r e l a t i v e l y little c o n t a m i n a t i o n b y m y e l i n o r m i c r o s o m a l m a t e r i a l . T h e s y n a p t o s o m e s s o m e t i m e s h a v e w h a t a p p e a r to b e fragm e n t s o f d e n d r i t e s o r d e n d r i t e s p i n e s a d h e r i n g to t h e m , a n d o t h e r profiles may represent dendritic fragments free of synaptic attachments in t h e p l a n e o f s e c t i o n . T h i s f r a c t i o n r e p r e s e n t s o n l y a b o u t 23% o f t h e t o t a l r e c o v e r e d p r o t e i n , y e t c o n t a i n s , o n t h e a v e r a g e , 43% o f the recovered acetylcholine. Thus the relative specific concentration of a c e t y l c h o l i n e in t h e f r a c t i o n is w e l l a b o v e u n i t y , i n d i c a t i n g a c o n s i d e r a b l e e n r i c h m e n t o f c h o l i n e r g i c t e r m i n a l s in this f r a c t i o n . T h e a c e t y l c h o line c o n t e n t o f t h e h o m o g e n a t e s w a s n o t s i g n i f i c a n t l y d i f f e r e n t f r o m t h a t o f w h o l e t i s s u e ( T a b l e I), s h o w i n g t h a t t h e l a t t e r d o e s n o t c o n t a i n f r e e
88
E . J . SIMON ET AL.
87, ,,..,
O'9
O
e"
~,
>~'~
t9
ELASMOBRANCH
89
SYNAPTOSOMES
acetylcholine. This indicates that as in mammalian caudate nucleus (12) cholinergic neurons are represented mainly by their terminals and that synaptosome formation has occurred in high yield with little loss of transmitter (for discussion see ref. 13). Lactate dehydrogenase, as would be expected of a soluble cytoplasmic marker, is mainly recovered in fraction $2; however, a significant proportion occurs in fraction P2L where it no doubt represents soluble cytoplasm sequestered within synaptosomes. Significantly, this fraction of lactate dehydrogenase is largely occluded (Table I) in contrast to that in the supernatant (Sz) fraction, which is almost entirely free. Somewhat puzzling, initially, was the considerable amount of acetylcholine found in fraction $2. Since the fraction contains acetylcholinesterase [the activity using acetylthiocholine as substrate (14) was 0.8 /~mol/mg of protein/min, about half that of mammalian cortex] and no anticholinesterase was added, this acetylcholine cannot be free: electron microscopy of the pellet (Figure 2) obtained when this fraction was fixed with glutaraldehyde and sedimented at 50,000g for 1 hr revealed the presence of synaptosomes heavily contaminated with membrane fragments. The external membranes of these synaptosomes often showed evidence of blebbing or discontinuities. We therefore conclude that the acetylcholine of this fraction is associated with incompletely sealed or partially disrupted synaptosomes. Such structures would not show latency or occlusion with respect to their content of lactate dehydrogenase or other soluble enzymes and would rapidly equilibrate with 0.8 M sucrose, thus losing their bouyancy, the property necessary for flotation and pellicle formation. Difficulties were encountered in measuring fumarase levels which were low in all fractions under the assay conditions used. The distribu-
T A B L E II UPTAKE OF CHOLINE BY DOGFISH OLFACTORY LOBE SYNAPTOSOMES a U p t a k e o f choline (pmol/min/g tissue) at 24~ in Suspension medium Tris-buffered s e a water E l a s m o b r a n c h Ringer
Fraction P2L 16.4 --
Fraction P2L-fast b 8.1 13.6
Fraction S2-fast ~ -0
Rates o f uptake refer to a 10-min incubation period. b In t h e s e e x p e r i m e n t s , the h o m o g e n a t e was centrifuged immediately at 17,000g for 1 hr to form a combined P1 + P~H pellet.
90
E. J. S I M O N E T AL.
=o_
ELASMOBRANCH SYNAPTOSOMES
91
tion indicates that fraction PzH, as in the squid, is enriched in mitochondria. However, electron micrography, while confirming this, showed the presence of not inconsiderable numbers of synaptosomal profiles and membrane fragments some of which were apparently of synaptosomal origin. Choline Uptake. Uptake of choline by fraction P~L was linear for the first 10 rain and thereafter showed some fall in rate. Initial rates are shown in Table II. These are comparable to those observed with mammalian synaptosome preparations of similar acetylcholine content (e.g., rabbit cerebellum 24 pmol/min/g at 25~ M.J. Dowdall, personal communication). Osmotic shock caused 90-100% inhibition and hemicholinium (100/zM), 50-90% inhibition. Noteworthy was the complete lack of uptake by fraction Sz, confirming the impression that the synaptosomes in this fraction are incompletely sealed. A kinetic analysis of the uptake of choline by fraction P2L will be required to determine whether this fraction contains the high-affinity uptake system specific for cholinergic synaptosomes (3, 15, 16), as well as the more widely distributed low-affinity system. However, at the choline concentration used (1.0 /zM), uptake would occur equally via both systems if dogfish synaptosomes are similar to others examined. A preparation of synaptosomes from a vertebrate obtained in a single step by a relatively rapid procedure which apparently selects well-sealed structures may have advantages in the study of metabolic processes, especially those dependent on labile components.
ACKNOWLEDGMENTS We thank Joe Keeter, Deparment of Neuroscience, Albert Einstein College of Medicine, for electron microscopy of fraction $2. This work was supported by grant No. DA 00017 from the National Institute on Drug Abuse. E.J.S. is a career scientist of the Health Research Council of the City of New York.
REFERENCES 1. DOWDALL,M.J., and WHITTAKER,V.P. 1973. Comparative studies on synaptosome formation: The preparation of synaptosomes from the head ganglion of the squid,
Loligo pealii. J. Neurochem. 20:921-935. 2. NEWKIRK, R.F., BALLOU, E.W., VICKERS, G., and WHITTAKER, V.P. 1976. Comparative studies in synaptosome formation: Preparation of synaptosomes from the ventral nerve cord of the lobster (Homarus americanus). Brain Res. 101:103-111.
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3. DOWDALL, M.J., and SIMON, E.J. 1973. Comparative studies on synaptosomes: 4.
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10. 11. 12.
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Uptake of [N-Me-3H]choline by synaptosomes from squid optic lobes. J. Neurochem. 21:969-982. POLLARD, H.B., BOHR, V.A., DOWDALL, M.J., and WHITTAKER, V.P- 1973. A chlorpromazine-sensitive, high affinity uptake system for L-noradrenaline in synaptosomes from the optic lobe of squid. Biol. Bull. (Woods Hole) 145:449-450. HERN/~NDEZ, A.G., LANGFORD, G.M., MARTINEZ, J.L., and DOWDAEL, M.J. 1976. Protein synthesis by synaptosomes from the head ganglion of the squid, Lotigo pealii (submitted for publication). SIMON, E.J., HILLER, J.M., and EDELMAN, I. 1973. Solubilizafion of a stereospecific [aH]etomorphine-macromolecular complex from a rat brain membrane fraction. Proc. Natl. Acad. Sci., U.S.A. 70:1947-1949. WHITTAKER, V.P., and GREENGARD,P. 1971. The isolation of synaptosomes from the brain of a teleost fish, (Centriopristes striatus). J. Neurochem. 18:173-176. MACINTOSH, F.C., and PERRY, W.L.M. 1961. Biological estimation of acety!choline. Methods Med. Res. 3:78-92. WHITTAKER, V.P., and BARKER, L.A. 1972. The subcellular fractionation of brain tissue with special reference to the preparation of synaptosomes and their component organelles. Pages 1-52, in FRIED, R., (ed.), Methods in Neurochemistry, Vol. 2, Marcel Dekker, New York. MILLONIG, G . 1961. Advantages of a phosphate buffer for osmium tetroxide solutions in fixation. J. Appl. Phys. 32: 1637. REYNOLDS, E.S. 1963. The use of lead citrate at high pH as an electron-opaque stair in electron microscopy. J. Cell Biol. 17:208-212. LAVERTY,R., MICHAELSON,I.A., SHARMAN,D. F., and WHITTAKER, V.P. 1963. The subcellular localization of dopamine and acetylcholine in the dog candate nucleus. Br. J. Pharmacol. 21:482-490. WHITTAKER, V.P. 1971. Subcellular localization of neurotransmitters, Adv. Cytopharmacol. 1:319-330. ELLMAN, G.L., COURTNEY, K.D., ANDRES, V., and FEATHERSTONE,R.M. 1961. A new and rapid colorimetric determination of acetylcholinesterase activity, Biochem. Pharmacol. 7:88-95. DOWDALL, M.J., WACHTEER, K., and HENDERSON, F. 1975. Comparative studies on the high affinity choline uptake system. Abstr. 5th Int. Meet. Int. Soc. Neurochem., Barcelona, p. 122. DOWDALL, M.J. 1976. Synthesis and storage of acetylcholine in cholinergic nerve terminals. Pages 585-607, in BERL, S., CLARKE, D.D., and SCHNEIDER, O. (eds.), Metabolic Compartmentation in the Brain, Plenum Press, New York.
