Neurotransmitter-Specific Synaptosomes in Rat Corpus Striatum: Morphological Variations*

Proceedings of the National Academy of Sciences Vol. 68, No. 1, pp. 155-159, January 1971

EDUARD GFELLER*, MICHAEL J. KUHARt, AND SOLOMON H. SNYDERtj Departments of *Anatomy, of tPharmacology and Experimental Therapeutics, and of TPsychiatry and Behavioral Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

Communicated by David Bodian, October 28, 1970

ABSTRACT This communication describes ultra- mossy fiber endings have been separated from smaller synap structural variations among synaptosomal fractions tosomes in the cerebellum; however, both populations are rich isolated from the corpus striatum of the rat by incomplete equilibrium sedimentation in sucrose density gradients, in acetylcholine (7). Cotman et al. (8) also reported morpho- and attempts to relate the variations to neurotransmitter- logical differences in synaptosomal populations separated by specific synaptosomes. The concentration of synapto- differential centrifugation. Recently (9), various neurotrans- somes in each fraction of the density gradient was found mitters in brain tissue were labeled with different radioactive to be correlated with the concentration of potassium, a variations in the sedimentation marker for cytoplasm occluded within synaptosomes. isotopes, and subtle properties Monoamine oxidase activity was found to be correlated of synaptosomes that store different neurotransmitters were with the incidence of free mitochondria in the gradients. thereby discriminated. Using this approach and centrifuging Synaptosomes from denser gradient fractions showed a sucrose density gradients for brief intervals (a procedure desig- markedly increased frequency of adherent postsynaptic nated "incomplete equilibrium sedimentation"), we have elements and intraterminal mitochondria. These denser gradient fractions were rich in synaptosomes containing been able to enhance the resolution of these synaptosomes norepinephrine, dopamine, serotonin, and acetylcholine, (10, 11). while synaptosomes in lighter portions of the gradients We describe here the ultrastructural variations among sub- were rich in -y-aminobutyric acid and other amino acids. cellular fractions isolated from the corpus striatum of the rat These data suggest that significant morphological vari- to ations may exist among different neurotransmitter- by incomplete equilibrium sedimentation, and attempt specific nerve terminals in the brain. correlate them with the neurotransmitter-specific synaptosomes contained in different subcellular fractions. Ascertaining the function of morphologically identifiable syn- METHODS AND MATERIALS apses in the brain is a fundamental problem of neurobiology. Adult male rats (Sprague-Dawley, 150-200 g) were killed by A knowledge of which synapses utilize which neurotransmit- decapitation and their brains were rapidly removed. The ters would constitute a great advance toward an understand- striatum, weighing approximately 100 mg, was excised ac- ing of synaptic organization in the brain. Subcellular fraction- cording to the method of Glowinski and Iversen (12). ation of brain has been a useful approach to elucidating When examining the subcellular distribution of 5-hydroxy- chemical features of ultrastructural elements. When brain tryptamine (5-HT), norepinephrine (NE), or GABA, the tissue is homogenized in isotonic sucrose, large numbers of striatal tissue was incubated in 2 ml of modified Krebs-Hense- nerve terminals "pinch off" to form intact, membrane-bound leit (13) bicarbonate medium, with glucose and one-half the particles called "synaptosomes," which can be isolated by original concentration of calcium, containing radiolabeled differential and density gradient centrifugation (1, 2). Such 5-HT (5 X 10-8 M), NE (10-v M) or GABA (10-6 M). Brain- particles contain various putative transmitter substances, slices accumulate 5-HT (14-16), NE (17-19), or GABA (20- such as acetylcholine, norepinephrine, dopamine, serotonin, 22) by specific transport systems which appear to label the and 'y-aminobutyric acid (GABA) (1-3). Isolation of the endogenous pools. The dopamine-containing neurons of the "synaptosomal" fraction of brain tissue, however, does not corpus striatum accumulate both NE and dopamine (23, 24). provide information about the morphology of the nerve ter- The subcellular distribution of exogenous radiolabeled 5-HT, minals that store specific neurotransmitters. Several workers NE, and GABA in brain homogenates in sucrose gradients is were able to resolve partially cholinergic and noncholinergic identical to that of the endogenous compounds (11, 25, 3). synaptosomes (4), certain small synaptosomes containing We therefore utilized radioactive 5-HT, NE, or GABA to histamine (5), and serotonin-containing particles from those label the populations of synaptosomes containing the endog- containing acetylcholine (6). Large synaptosomes from enous compounds. Since the dopamine-containing neurons accumulated dopamine and NE by the same mechanism, Abbreviations: GABA, -y-aminobutyric acid; NE, norepi- either catecholamine can be used to label the striatal dopa- nephrine; 5-HT, 5-hydroxytryptamine. mine terminals, and in our experiments [,'H]NE was em- Requests for reprints may be addressed to Dr. Solomon H. ployed. When tissue was incubated with 5-HT, NE, or GABA, Snyder, Department of Pharmacology and Experimental Thera- either nialamide (a monoamine oxidase inhibitor) or amino- peutics, The Johns Hopkins University School of Medicine, oxyacetic acid (a GABA: glutamate transaminase inhibitor) Baltimore, Maryland 21205. was present (10-5 M) in the incubation medium to prevent 155 Downloaded by guest on September 25, 2021 156 Zoology: Gfeller et al. Proc. Nat. Acad. Sci. USA

or more vesicles. Free mitochondria were also counted in the electron z micrographs. Synaptosomes were classified according 0 to the presence of a synaptic cleft (postsynaptic element m (2 co-J attached) and according to the presence or absence of intra- -j terminal 0 mitochondria. The correlation coefficient between 0 H the ratings of the three observers of the five gradients was LL 0 0.92. Synaptosomal circumference was determined by means z z of a map measure. Preliminary studies have shown that cir- U U cumference correlates 0wXULI well with cut surface area (as deter- a. mined by weighing of cut-outs), although circumference in- creases more with size of the particle than with cut surface 2 3 4 5 6 7 8 9 10 (E. Gfeller, unpublished observations). Distribution of parti- FRACTION NUMBER cles within the pellet was quite homogeneous, apparently as FIG. 1. Composite figure showing separation of rat brain a result of resuspending the first pellet during the fixation synaptosomes containing serotonin (5-HT), norepinephrine procedure. Pellets of the three fractions in which synaptosomal (NE), and y-aminobutyric acid (GABA) in linear continuous characterstics were assessed quantitatively were of the same sucrose density gradients. The radioactivity profiles of exogenous size and contained approximately the same number of par- radiolabeled 5-HT, NE, and GABA were used as markers for ticles. different synaptosomal populations. Tissue slices were incubated with any two of these compounds, one "IC-labeled, the other RESULTS 3H-labeled. Separation of synaptosomes on sucrose density gradients Incomplete equilibrium sedimentation separated synapto- degradation of the original compounds. Under these condi- somes storing 5-HT and GABA with little overlap (Fig. 1). tions 90-100% of tissue radioactivity was associated with the The norepinephrine-storing synaptosomes had sedimentation original compound, as demonstrated by paper chromatog- characteristics similar to those of 5-HT, but were in slightly raphy. lighter portions of the gradients than the 5-HT containing Incubations were performed in a Dubnoff metabolic shaker particles. In repeated experiments using several different areas in a 95% 02-5% CO2 atmosphere at 37°C. of brains of rat, guinea pig, and hamster this difference was After incubation, the slices were removed from the medium, consistently observed (11). In other experiments, endogenous rinsed in fresh, ice-cold 0.32 M sucrose (Mallinckrodt Ana- acetylcholine assayed in these gradients was found in a popula- lytic Reagent Sucrose) and homogenized for about 1 min in 10 tion of particles whose pattern of distribution was near the vol of ice-cold 0.32 M sucrose in Potter-Elvehjem glass ho- peaks of norepinephrine and 5-HT (Kuhar, Snyder, and Sael- mogenizers fitted with a Teflon pestle (0.1-0.15 mm clearance). ens, unpublished observations). Recently we found that Crude mitochondrial pellets prepared by differential cen- histamine and the activity of the enzyme histamine methyl trifugation were centrifuged for brief periods (15 min at transferase occur in a population of synaptosomes with sedi- 100,000 X g) on linear continuous sucrose gradients (1.5-0.32 mentation properties similar to those containing acetylcholine M) and fractions were obtained as previously described (10). (Kuhar, Taylor, and Snyder, in preparation). In the measurement of gradient in potassium, fractions Correlation of potassium concentration with number of conical centrifuge tubes were immersed in boiling water for synaptosomes in density gradients 30 min and at for centrifuged 50,000 X g 10 min, and potas- The concentration of potassium has sium was measured in the supernatant fluids with a Techtron been used as a marker for atomic absorption spectrophotometer. Monoamine cytoplasm occluded within synaptosomes (2). If our sam- oxidase pling methods are the activity was determined in unboiled aliquots of gradient valid, concentration of synaptosomes the at different levels in the sucrose density gradient should corre- fractions by method of Wurtman and Axelrod (26). late with the For electron three consecutive distribution of potassium concentration, pro- microscopy, gradient frac- vided that the volume of synaptosomes in the different tions were pooled (10), the sucrose molarity was adjusted to popu- 0.5 M sucrose, and the samples were fixed with 1% KMnO4 in ice-cold 100 mM veronal acetate buffer (pH 7.4). After 20 H min at 4°C, the fractions were centrifuged at 30,000 X g for U5 20 + 10 min. The resulting were in -J pellets resuspended 0.5 ml of z 44 fixative and recentrifuged for 5 min at 13,000 X g in a Beck- (h 3 0 man Microfuge at 4°C. Resultant pellets were sectioned from 0 the tubes with a 10 razor-blade, dehydrated in acetone, and z2 H W embedded in Araldite. Sections were cut on Sorvall MT-1 and w MT-2B ultramicrotomes, stained with 1:100 lead hydroxide, W0 and viewed in a RCA EMU 3F electron microscope. Random WA. a. electronmicrographs were taken from sections from five differ- FRACTION NUMBER ent were gradients. Photographic prints (38,000X) judged FIG. 2. Distribution of intracullar potassium (K+) and by at least three independent observers. 238 electron micro- incidence of synaptosomes in sucrose density gradients. Fre- graphs were analyzed. Altogether, 1722 particleswere counted, quency of synaptosomes represents the average number (ISE) of which 464 -were classified as synaptosomes. A synaptosome of synaptosomes per 35 Mum2 in electron-microscopic samples of was defined as a membrane-bound particle containing three pellets from at least 18 samples from two gradients. Downloaded by guest on September 25, 2021 Vol. 68, 1971 Neurotransmitter-Specific Synaptosomes 157

lations does not vary greatly. The close correlation (Fig. 2) °Or 1.5 suggests that our quantitation of synaptosomes by electron

microscopy provide a reliable estimate of the incidence of In synaptosomes within gradient fractions, and shows that there is not an inordinate variation in synaptosomal volume over z 20 1.0 the density gradient. x Distributions of monoamine oxidase activity and of free c 2 mitochondria 0 Monoamine oxidase activity in brain tissue appears to be c lo I0.5 0~ localized in free mitochondria as well as in mitochondria I? within nerve terminals (2). When sucrose density gradients of brain particles are centrifuged to apparent density equilibrium, free mitochondria sediment in a denser area of the gradients than do synaptosomes (10). With incomplete equi- 2 3 4 5 6 7 8 9 10 librium sedimentation, the distributions of monoamine ox- FRACTION NUMBER idase activity and of free mitochondria (counted directly) FIG. 3. Distribution of ['3H]norepinephrine ('H-NE), mono- overlapped that of the synaptosomes determined by ['HINE amine oxidase activity (MAO), and incidence of free mito- marker (Fig. 3). chondria in sucrose density gradients. Frequency of free mito- Morphology of synaptosomes distributed over the density chondria represents the average number (±SE) of mitochondria gradient per 35 /m2 inl at least 18 samples from two gradients. The ultimate aim of studies directed toward sel)aration and purification of neurotransmitter-specific synaptosomes is to homogenized and subjected to the same subcellular fractiona- define morphological characteristics that are unique for each tion procedures as incubated brain slices. Fractions 3, 4, and class of synaptosomes. As a preliminary step in this en- 5 were examined under the electron microscope and the in- deavor, we compared several morphological features of syn- cidence of synaptosomes with intact clefts, intraterminal aptosomes in different fractions (Figs. 4 and 5). The circum- mitochondria, or both was assessed. The same relative dis- ferences of synaptosomes were compared in fractions 3, 4, and tribution of these elements was obtained as in slices of brain 5, as well as the incidence of synaptosomes with intact syn- incubated with radioactive amines. ap)tic clefts, with intraterminal mitochondria, and without In a morphological comparison of synaptosomes with either clefts or mitochondria. Synaptosomes with intact clefts nerve terminals in intact tissue, incubated brain slices were occurred much more frequently in denser fractions, ranging fixed and examined under the electron microscope. In a series from a 40% incidence in fraction 3 to less than 10% in fraction of 110 nerve terminals of rat corpus striatum about 39% con- 5. Since the average proportion of the surface area of syn- tained intraterminal mitochondria, which is similar to the aptosomes in the rat corpus striatum occupied by the synaptic total incidence (about 35%) of synaptosomes with intra- cleft is up to 35% (E. Gfeller, unpublished observations), it is terminal mitochondria in fractions 3, 4, and 5 of the density possible that almost all of the synaptosomes in fraction 3 gradients. This again suggests that nerve terminals in the actually have intact clefts. corpus striatum are not differentially altered during the prel)- The incidence of intraterminal mitochondria in fraction 3 aration of synaptosomes, and that synaptosomes may mirror was 50% greater than in fraction 4 and double that of fraction quantitatively important features of intact brain tissue. 5 (Fig. 4). Whittaker (27), using isopycnic centrifugal procedures, and Cotman et al. (8), using differential centrifuga- DISCUSSION tion, found that mitochondria-rich synaptosomes sediment In the l)resenlt study, a statistically significant greater in- more rapidly than those with fewer mitochondria. cidence of synap)tosomes with adherent postsynaptic elements Since size is an important factor determining the sedimen- as well as with intraterminal mitochonidria was observed in tation rate of particles, one might expect differences in syn- denser areas of the sucrose gradients. AMoreover, we were able aptosomal size throughout the gradient. However, when syn- to correlate the synaptosomal profiles waith concentration of aptosomal circumference was measured without including the potassium in gradients, as well as the incidence of mitochon- postsynaptic element, there was no difference between frac- dria with the distribution of monoamine oxidase activity (this tions 3, 4, and 5: respectively 1.90 i 0.07(SE) ,um(n = 52), enzyme being a marker for mitochondria). 1.89 ± 0.07 jam (n = 45), and 1.82 i 0.07 Mm(n = 90). But What determines the rate at which different synaptosomes since the incidence of intact synaptic clefts was greater in sediment through our sucrose density gradients? In experi- fraction 3 than in fractions 4 and 5, the synaptosomal circum- ments with differential centrifugation, larger particles sedi- ference including the postsynaptic element was greater in ment more rapidly (8). The giant mossy fiber endings of the fraction 3. Cotman et al. (8) also found that synaptosomes cerebellum are the most rapidly sedimenting synaptosomes, that sediment more rapidly have the larger diameters. In their appearing in "nuclear" fractions (7). "Microsomal" fractions work, the mean circumference of synaptosomes with clefts obtained by centrifuging post-mitochondrial supernatant (not including the cleft), 1.8 + 0.1 Am, did not differ from fluid at 20,000 X g for 30 min contain a large number of small those without clefts (1.7 ± 0.1 Mm). synaptosomes (5). By contrast, there is no difference in the It is conceivable that incubating brain slices may have diameter of synaptosomes from different regions of sucrose altered some morphological features. Accordingly, in some density gradients centrifuged to density equilibrium (27, 4). experiments fresh, unincubated rat corpus striatum was Our centrifugal techniques presumably embody features both Downloaded by guest on September 25, 2021 158 Zoology: Gfeller et al. Proc. Nat. Acad. Sci. USA

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ments ISE(n = total number of synaptosomal profiles). (Right) FIG. 5. Electron micrographs of synaptosomal profiles from fractions 3 and 5 and of synaptic profiles from rat striatum fixed in situ. Upper row: synaptosomes from fraction 5 (X31,000). Middle row: synaptosomes from fraction 3 (X31,000); adherent postsynaptic elements (clefts) and intraterminal mitochondria can be identified. Bottom row: rat striatum perfused with 2.5% glutaraldehyde after treatment with OS04 ( X20,000). 1, Gray's type I synaptic profiles; 2, synaptic profiles containing mitochondria; X, synaptic profile of Gray's type I with intraterminal mitochondrion; 4, synaptic profiles containing synaptic vesicles but no mitochondria and not forming membrane specializations, Bar = 1 ,um.

of sedimentation rate and density equilibrium. Although characteristics, namely the frequency of adherent postsyn- synaptosomal circumference did not differ in the different aptic elements and of intraterminal mitochondria. This sug- parts of the gradient, particles with adherent postsynaptic gests that significant morphological variations may exist elements occurred most frequently in the dense regions of the among different neurotransmitter-specific nerve terminals in gradient, so that total particle diameter in these denser re- the brain. If this is so it might ultimately be possible to iden- gions was somewhat greater than in the lighter portions of the tify the neurotransmitters utilized by given nerve terminals gradients. solely on the basis of morphological appearance in intact Our centrifugal procedures did discriminate between syn- brain tissue. However, it is possible that various effects of aptosomes on the basis of the incidence of intraterminal mito- homogenization may produce some of the morphologic differ- chondria and adherent postsynaptic membranes. Density ences. equilibrium centrifugation of sucrose gradients has met with It is difficult to ascertain what proportion of the synapto- limited success in resolving synaptosomes on the basis of somes in different gradient fractions contain particular neuro- intraterminal mitochondria or frequency of postsynaptic transmitters. Fraction 3, representing the 5-HT and dopamine elements (27). Thus, our methods differ from density equi- terminals, contains about 20% of all the synaptosomes in the librium procedures in the efficiency in separating neurotrans- gradient, about half of the gradient's 5-HT and and cat- mitter-specific synaptosomes and in differentiating popula- echolamine, but only a small percentage of the GABA in the tions of synaptosomes on the basis of specific morphological gradient. H6kfelt (28) estimated that about 15% of the Downloaded by guest on September 25, 2021 Vol.V68,619711NeurotraIismitter-Specific Synaptosomes 159

nerve terminals in the rat corpus striatum are dopaminergic. 8. Cotman, C., D. H. Brown, B. W. Harrell, and N. G. If a similar proportion are serotonergic, it would seem that a Anderson, Arch. Biochem. Biophys., 136, 436 (1970). 9. Iversen, L. L., and S. H. Snyder, Nature (Lond.), 220, 796 large proportion of the synaptosomes in fraction 3 might con- (1968). tain one or the other of these two putative neurotransmitters. 10. Kuhar, M. J., A. I. Green, S. H. Snyder, and E. Gfeller, Acetylcholine- and histamine-containing synaptosomes are Brain Res., 21, 405 (1970). also localized primarily in fractions 3 and 4 (Kuhar, 11. Kuhar, M. J., E. Shaskan, and S. H. Snyder, J. Neuro- Taylor, Snyder, and Saelens, unpublished results). chem., in press (1970). 12. Glowinski, J., and L. L. Iversen, J. Neurochem., 13, 655 Glutamic and aspartic acids have been postulated to be (1966). major excitatory neurotransmitters in the brain (29, 30). 13. Krebs, H. A., and K. Henseleit, Hoppe-Seyler's Z. Recently, we found that when brain slices were incubated Physiol. Chem., 210, 33 (1932). with a large number of radiolabeled amino acids, glutamic 14. Blackburn, K. J., P. C. French, and R. M. Merrills, Life and Sci., 6, 1653 (1967). aspartic acid were localized in a population of synapto- 15. Shaskan, E. G., and S. H. Snyder, J. Pharmacol. Exp. somes discriminable both from those storing other "non- Ther., 175, 404 (1970). transmitter" amino acids and from the GABA-containing 16. Shaskan, E. G., S. H. Snyder, and E. D. Hendley, Fed. particles (Wofsey, Kuhar, Snyder, in preparation). Thus, with Proc. 29, 263 (1970). our procedures the distribution of amines such as acetyl- 17. Dengler, J. J., I. A. Michaelson, H. E. Spiegel, and E. Titus, Int. J. Neuropharmacol., 1, 23 (1962). choline, 5-HT, catecholamines, and histamine has a maximum 18. Snyder, S. H., A. I. Green, and E. D. Hendley, J. Phar- in a relatively dense area of the gradient, while most of the macol. Exp. Ther., 164, 90 (1968). particles storing amino acids such as GABA, glutamic acid 19. Snyder, S. H., A. Green, E. D. Hendley, and E. Gfeller, and aspartic acid cluster in a less dense region of the gradients. Nature (London) 218, 174 (1968). 20. Iversen, L. L., and M. J. Neal, J. Neurochem., 15, 1141 This work was supported by USPHS Grants I\IH-18501, 1- (1968). RO1-NB-07275, and NB-07934. Solomon H. Snyder is a re- 21. Kuriyama, K., E. Roberts, and J. Vos, Brain Res., 9, 231 cipient of NINMH Research Scientist Development Award K3- (1968). MH-33128. 22. Roberts, E., and K. Kuriyama, Brain Res., 8, 1 (1968). 23. Snyder, S. H., and J. T. Coyle, J. Pharmacol. Exp. Ther.; 1. De Robertis, E., Science, 156, 907 (1967). 165, 78 (1969). 2. Whittaker, V. P., Progr. Biophys. Mol. Biol., 15, 41 24. Coyle, J. T., and S. H. Snyder, J. Pharmacol. Exp. Ther., (1965). 170, 221 (1969). 3. Neal, M. J., and L. L. Iversen, J. Neurochem., 16, 1245 25. Green, A. I., S. H. Snyder, and L. L. Iversen, J. Pharma- (1969). col. Exp. Ther., 168, 264 (1969). 4. De Robertis, E., A. Pellegrino De Iraldi, G. Rodriguez De 26. Wurtman, R. J., and J. Axelrod, Biochem. Pharmacol., 12, Lores Arnaiz, and L. Salganicoff, J. Neurochem., 9, 23 (1962). 1439 (1963). 5. Kataoka, K., and E. De Robertis, J. Pharmacol. Exp. 27. Whittaker, V. P., Biochem. J., 106, 412 (1968). Ther., 156, 114 (1967). 28. Hokfelt, T., Z. Zellforsch. Mikroskop. Anat., 91, 1 (1968). 6. Michaelson, I. A., and V. P. Whittaker, Biochem. Phar- 29. Curtis, D. R., J. W. Phillis, and J. C. Watkins, J. Physiol. macol., 12, 203 (1963). (London), 150, 656 (1960). 7. Israel, M., and V. P. Whittaker, Experientia, 21, 325 30. Krnjevic, K., and J. W. Phillis, J. Physiol. (London), 165, (1965). 274 (1963). Downloaded by guest on September 25, 2021