Immunocytochemical localization of S-100 protein binding sites in synaptosomal fractions and subfractions
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Synaptosome
Divalent
Postsynaptic density
Synaptic membrane
Free nerve ending
Cerebral ischemia was induced by permanently occluding the distal middle cerebral artery in healthy SD rats, and electronic microscope was used. After cerebral ischemia, the number of the synaptic terminals decreased and the synaptic structure was damaged; the amount of synaptic vesicles were decreased; the cristae of mitochondria were broken, dissolved and disappeared. Presynaptic and postsynaptic membrane became unclear; the outline of presynaptic element was irregular. Fourty-eight hours after ischemia, synaptic vesicles decreased so much that some disappeared. The mitochondria had vacuolar degeneration and sometimes disappeared. The presynaptic and postsynaptic membrane were damaged so seriously that the typical synaptic structure did not exist. After cerebral ischemia, the number of the synaptic terminals decrease and the synaptic structure are damaged.
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A rapid and simple method is described for separation of intact synaptosomes, synaptic plasma membranes and vesicles. Two synaptosome fractions were obtained by modified differential centrifugation. The rate zonal zentrifugation in a linear sucrose gradient (very low density) is suitable to obtain fractions highly enriched in synaptic plasma membranes and vesicles. Examination of the prepared fractions was done by enzyme marker activities and electron microscopy
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Differential centrifugation
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Density gradient
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Synaptosome
Free nerve ending
Synaptic membrane
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The human brain is estimated to contain trillions of synaptic nerve terminals. These are the connections between neurons that are responsible for transmitting information and are modified as a result of learning. A valuable tool for studying synapses is the isolated nerve terminal, or synaptosome, which is obtained by homogenizing the brain in such a way that individual synapses pinch off to form metabolically active compartments that can recapitulate neurotransmitter release. This protocol describes the stepwise fractionation of rat brain tissue to yield synaptosomes and synaptic vesicles, which can be used in many different experimental approaches to study the structure and protein composition of the synapse and even dissect the molecular mechanisms of neurotransmission.
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Free nerve ending
Synaptic membrane
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Synaptosome
Divalent
Postsynaptic density
Synaptic membrane
Free nerve ending
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Unique Lipid Chemistry of Synaptic Vesicle and Synaptosome Membrane Revealed Using Mass Spectrometry
Synaptic vesicles measuring 30-50 nm in diameter containing neurotransmitters either completely collapse at the presynaptic membrane or dock and transiently fuse at the base of specialized 15 nm cup-shaped lipoprotein structures called porosomes at the presynaptic membrane of synaptosomes to release neurotransmitters. Recent study reports the unique composition of major lipids associated with neuronal porosomes. Given that lipids greatly influence the association and functions of membrane proteins, differences in lipid composition of synaptic vesicle and the synaptosome membrane was hypothesized. To test this hypothesis, the lipidome of isolated synaptosome, synaptosome membrane, and synaptic vesicle preparation were determined by using mass spectrometry in the current study. Results from the study demonstrate the enriched presence of triacyl glycerols and sphingomyelins in synaptic vesicles, as opposed to the enriched presence of phospholipids in the synaptosome membrane fraction, reflecting on the tight regulation of nerve cells in compartmentalization of membrane lipids at the nerve terminal.
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Lipidome
SNAP25
Compartmentalization (fire protection)
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In this paper, our protocol for preparation of brain synaptosomes is described. Synaptosomes are a valuable model system for analysis of structural components of the synapse as well as for investigation of synaptic function. Synaptosomal preparations are necessary for understanding molecular changes at synapses where critical post-translational modifications of synaptic proteins may occur. Not only are synaptosomes rich in synaptic proteins, but they can be used for analyzing uptake of neurotransmitters into synaptic vesicles and for analysis of the involvement of neurotransmitter synthesis and release. Synaptosomes can be stimulated with increased calcium influx to release neurotransmitters. Synaptosomal preparations have been used in characterizing calcium dependent phosphorylation and activation of the GABA synthesizing enzyme GAD65 (L-glutamic acid decarboxylase with molecular weight of 65 kDa). By examining protein complexes on the membrane of synaptic vesicles obtained from synaptosomal preparations, it was possible to characterize the role of GAD65 in the coupled synthesis and vesicular uptake of GABA (γ-aminobutyric acid) culminating in GABA vesicular release, which contributes in an important way to fine-tuning of GABAergic neurotransmission.
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Ethanol was shown to cause a redistribution of synaptic vesicles in incubated synaptosomes. While the number of synaptosomes containing synaptic vesicles attached to the presynaptic membrane decreased markedly, an increase in the number of synaptosomes lacking membrane-vesicle associations was observed. The findings support the possibility of a presynaptic action of ethanol and point to the role of membrane-attached vesicles in synaptic transmission.
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