Separation of synaptosomes storing catecholamines and gamma-aminobutyric acid in rat corpus striatum

Kuhar, Michael J.; Green, Alan I.; Snyder, Solomon H.; Gfeller, Eduard

doi:10.1016/0006-8993(70)90420-8

Cited by 60 publications

(20 citation statements)

References 19 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Gradients were maintained at 40 for 12 hr before use. The gradients were centrifuged at 100,000 X g for 15 min with a SW 50.1 rotor in a Spinco L2-65B ultracentrifuge, a condition of "incomplete sedimentation equilibrium" (16,17). Fractions were obtained by piercing the bottoms of the tubes and collecting 10-drop aliquots.…”

Section: Methodsmentioning

confidence: 99%

“…Fractions were obtained by piercing the bottoms of the tubes and collecting 10-drop aliquots. 32 (19) as described (17). Monoamine oxidase was used as a marker for mitochondria because the sensitivity of the available radiometric assay (19) from each of these samples was analyzed for K+ content on a Techtron atomic absorption spectrophotometer.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Specific Glycine-Accumulating Synaptosomes in the Spinal Cord of Rats

Arregui

Logan

Bennett

et al. 1972

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

Subcellular fractionation of rat spinal cord on continuous sucrose density gradients provides evidence for the existence of a specific synaptosomal fraction (enriched in pinched-off nerve endings) that accumulates glycine selectively by way of a high-affinity transport system. The particles in this fraction sediment to a less-dense portion of sucrose gradients than do particles that accumulate neutral, basic, aromatic, and acidic amino acids. Particles accumulating y-aminobutyric acid are even less-dense than those storing exogenous glycine. The glycine-specific synaptosomal fraction also exists in the brain stem but not in the cerebral cortex. These findings provide neurochemical support for the suggestion that glycine has a specialized synaptic function, perhaps as a neurotransmitter, in mammalian spinal cord.Several lines of evidence favor a synaptic role for glycine in mammalian spinal cord. Most importantly, in neurophysiologic studies, glycine mimics the behavior of the natural inhibitory transmitter in its conductance effects and blockade by strychnine (1-3). The regional localization of glycine within the spinal cord is consistent with that of the natural inhibitory transmitter (4-7). Electron microscopic autoradiography reveals a localization of accumulated radioactive glycine predominantly in nerve terminals in the spinal cord (8, 9). Accumulated radioactive glycine can be released from the spinal cord by electrical depolarization of spinal cord slices (10, 11).Recently a high-affinity uptake system for glycine into slices (12,13) or synaptosomal preparations (13-15) of rat spinal cord was described that was not manifested by most other amino acids (14,15) and that was present in spinal cord (13-15) and brain stem (13), but not in the cerebral cortex (13-15).Previously we reported a synaptosomal fraction with distinct sedimentation characteristics on density gradient centrifugation of cerebral cortexes that accumulated glutamic and aspartic acids (16) by means of a high-affinity transport system. Nerve terminals in the spinal cord that accumulate radioactive glycine differ in their morphology from those that did not selectively take up this amino acid (9). Accordingly, it is conceivable that glycine-accumulating synaptosomes may differ in their physical properties from the general population of synaptosomes in the spinal cord. We report here that radiolabeled glycine is accumulated by homogenates of rat spinal cord into a unique synaptosomal fraction that can be distinguished from the particles that store other amino acids. METHODS AND MATERIALSSprague-Dawley male rats (150-200 g) were decapitated, and their spinal cords, medulla-pons, or cerebral cortexes rapidly removed and placed on dental wax on ice-. All subsequent procedures were conducted at 0-4°. After they were weighed, the tissues were homogenized in 20: 1 (v/w) 0.32 M sucrose with a glass homogenizer with a motor-driven Teflon pestle (0.09-0.11 mm clearance). The homogenates were centrifuged at 1000 X g for 10 min to remove nuclei an...

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Specific Glycine-Accumulating Synaptosomes in the Spinal Cord of Rats

Arregui

Logan

Bennett

et al. 1972

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

show abstract

“…Correlation of potassium concentration with number of synaptosomes in density gradients The concentration of potassium has been used as a marker for cytoplasm occluded within synaptosomes (2 (10). With incomplete equilibrium sedimentation, the distributions of monoamine oxidase activity and of free mitochondria (counted directly) overlapped that of the synaptosomes determined by ['HINE marker (Fig.…”

Section: Methodsmentioning

confidence: 98%

“…Crude mitochondrial pellets prepared by differential centrifugation were centrifuged for brief periods (15 min at 100,000 X g) on linear continuous sucrose gradients (1.5-0.32 M) and fractions were obtained as previously described (10).…”

Section: Methodsmentioning

confidence: 99%

“…Recently (9), various neurotransmitters in brain tissue were labeled with different radioactive isotopes, and subtle variations in the sedimentation properties of synaptosomes that store different neurotransmitters were thereby discriminated. Using this approach and centrifuging sucrose density gradients for brief intervals (a procedure designated "incomplete equilibrium sedimentation"), we have been able to enhance the resolution of these synaptosomes (10,11).…”

mentioning

confidence: 99%

See 1 more Smart Citation

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

Gfeller

Kuhar

Snyder

1971

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

This communication describes ultrastructural variations among synaptosomal fractions isolated from the corpus striatum of the rat by incomplete equilibrium sedimentation in sucrose density gradients, and attempts to relate the variations to neurotransmitterspecific synaptosomes. The concentration of synaptosomes in each fraction of the density gradient was found to be correlated with the concentration of potassium, a marker for cytoplasm occluded within synaptosomes. Monoamine oxidase activity was found to be correlated with the incidence of free mitochondria in the gradients.Synaptosomes from denser gradient fractions showed a markedly increased frequency of adherent postsynaptic elements and intraterminal mitochondria. These denser gradient fractions were rich in synaptosomes containing norepinephrine, dopamine, serotonin, and acetylcholine, while synaptosomes in lighter portions of the gradients were rich in -y-aminobutyric acid and other amino acids. These data suggest that significant morphological variations may exist among different neurotransmitterspecific nerve terminals in the brain. mossy fiber endings have been separated from smaller synap tosomes in the cerebellum; however, both populations are rich in acetylcholine (7). Cotman et al. (8) also reported morphological differences in synaptosomal populations separated by differential centrifugation. Recently (9), various neurotransmitters in brain tissue were labeled with different radioactive isotopes, and subtle variations in the sedimentation properties of synaptosomes that store different neurotransmitters were thereby discriminated. Using this approach and centrifuging sucrose density gradients for brief intervals (a procedure designated "incomplete equilibrium sedimentation"), we have been able to enhance the resolution of these synaptosomes (10, 11).We describe here the ultrastructural variations among subcellular fractions isolated from the corpus striatum of the rat by incomplete equilibrium sedimentation, and attempt to correlate them with the neurotransmitter-specific synaptosomes contained in different subcellular fractions. Ascertaining the function of morphologically identifiable synapses in the brain is a fundamental problem of neurobiology. A knowledge of which synapses utilize which neurotransmitters would constitute a great advance toward an understanding of synaptic organization in the brain. Subcellular fractionation of brain has been a useful approach to elucidating chemical features of ultrastructural elements. When brain tissue is homogenized in isotonic sucrose, large numbers of nerve terminals "pinch off" to form intact, membrane-bound particles called "synaptosomes," which can be isolated by differential and density gradient centrifugation (1, 2). Such particles contain various putative transmitter substances, such as acetylcholine, norepinephrine, dopamine, serotonin, and 'y-aminobutyric acid (GABA) (1-3). Isolation of the "synaptosomal" fraction of brain tissue, however, does not provide information about the morphol...

show abstract

Ultrastructural characterization of synaptosomes from neonatal and adult rats with special reference to monoamines

Kanerva

Tissari

Suurhasko

et al. 1977

J of Comparative Neurology

View full text Add to dashboard Cite

The crude mitochondria1 fraction P2 and subfractions of it were prepared from the brain stem, hemispheres, hypothalamus and striatum of adult rats and from the hemispheres and brain stem or diencephalon-mesencephalon and pons-medulla oblongata of 1-day-old rats. Samples were fixed in glutaraldehyde-osmium, KMnO, or by Tranzer's triple fixation method (aldehydes-chromate-dichromate-osmium) and examined by electron microscopy. With all these fixation techniques 80-95% of processes in the adult samples were found to be mature synaptosomes. After incubation with either 10-100 pg/ml of 5-hydroxydopamine or a-methylnoradrenaline, and after permanganate or triple fixation, monoamine synaptosomes, containing small granular vesicles were detected.They were found in all the brain regions studied and in the fraction P2 and subfractions C and D but only a t a frequency of less than 1%. In samples from l-dayold rats using the different fixatives, synaptosomes comprised 10-30% of all processes. Nerve endings were a t very different stages of maturation in sharp contrast to the rather uniform type of adult synaptosomes. They varied from processes still without any substructures to mature synaptosomes containing synaptic junctions. Monoamine synaptosomes containing small granular vesicles were detected after amine incubation and KMnO, or triple fixation. Their frequency was lower than that in the adult. Most common were very immature terminals containing smooth endoplasmic reticulum and a very few small granular vesicles, indicating a very early appearance of the capacity to form small granular vesicles under experimental conditions. The immature terminals had all the characteristics of the growth cone and might partly have originated from growth cones a t the tips of growing axons and dendrites. Less frequent were more mature synaptosomes containing a greater number of small granular vesicles and no longer any smooth endoplasmic reticulum. The crude microsomal fraction P3 was also examined in the adult brain regions and the brain stem of 1-day-olds.This fraction contained a small number of small agranular vesicles originating from cell bodies and nonterminal axons; their number was smaller in 1-day-old than in adult rats. The present results are in agreement with the high proportion of immature monoamine terminals in newborn rat brain detected earlier by fluorescence histochemistry.Earlier studies have demonstrated that density, contrasting to the rather uniform synaptosomes in a low proportion can be iso-adult type of synaptosomes (Spence and lated from the brain of newborn rats.

show abstract

Separation of synaptosomes storing catecholamines and gamma-aminobutyric acid in rat corpus striatum

Cited by 60 publications

References 19 publications

Specific Glycine-Accumulating Synaptosomes in the Spinal Cord of Rats

Specific Glycine-Accumulating Synaptosomes in the Spinal Cord of Rats

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

Ultrastructural characterization of synaptosomes from neonatal and adult rats with special reference to monoamines

Contact Info

Product

Resources

About

Separation of synaptosomes storing catecholamines and gamma-aminobutyric acid in rat corpus striatum

Cited by 60 publications

References 19 publications

Specific Glycine-Accumulating Synaptosomes in the Spinal Cord of Rats

Specific Glycine-Accumulating Synaptosomes in the Spinal Cord of Rats

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

Ultrastructural characterization of synaptosomes from neonatal and adult rats with special reference to monoamines

Contact Info

Product

Resources

About

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