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

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