The effect of ethanol on the distribution of synaptic vesicles in cortical synaptosomes of the rat.
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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.Keywords:
Synaptosome
Synaptic membrane
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Neurotransmission requires the release of neurotransmitters from synaptic vesicles. This occurs via fusion of the vesicle to the pre-synaptic membrane upon stimulation. However, not all synaptic vesicles are equally releasable, and it has long been debated why the majority of synaptic vesicles do not respond to physiological levels of stimulation. I demonstrate here, using live-cell antibody-tagging in rat hippocampal cultures, that only young synaptic vesicles are releasing neurotransmitter, and that they become more reluctant to release as they age. This inactivation of synaptic vesicles is not strictly an ageing-dependent process, but conditional upon vesicle usage. I report here that synaptic vesicles release ~260 times, on average, before becoming inactive, and that increasing usage frequency speeds up inactivation. The inactivation is caused by contamination of synaptic vesicles with the cell membrane protein SNAP25. SNAP25 can interact with the vesicle protein CSPα in cis- complexes on the vesicle itself. This sequesters CSPα and prevents the formation of trans- complexes with SNAP25 on the cell membrane. This trans-interaction, however, promotes vesicle fusion to the cell membrane. The more often a vesicle has fused to the cell membrane, the higher its chance is to be contaminated with SNAP25, and the less competent it is for future rounds of release. The inactivation of ageing synaptic vesicles is presumably coupled to usage to remove potentially damaged synaptic vesicles from neurotransmission. This hypothesis is strengthened by the observation of endocytosis defects and neurite degeneration when aged vesicles are forced to release. I further provide several timing parameters for key events in the life of synaptic vesicles, which can serve as a framework towards a quantitative model of the synaptic vesicle life cycle.
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Secretory Vesicle
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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
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Neurotransmission requires the release of neurotransmitters from synaptic vesicles. This
occurs via fusion of the vesicle to the pre-synaptic membrane upon stimulation. However, not
all synaptic vesicles are equally releasable, and it has long been debated why the majority of
synaptic vesicles do not respond to physiological levels of stimulation. I demonstrate here,
using live-cell antibody-tagging in rat hippocampal cultures, that only young synaptic vesicles
are releasing neurotransmitter, and that they become more reluctant to release as they age.
This inactivation of synaptic vesicles is not strictly an ageing-dependent process, but
conditional upon vesicle usage. I report here that synaptic vesicles release ~260 times, on
average, before becoming inactive, and that increasing usage frequency speeds up
inactivation. The inactivation is caused by contamination of synaptic vesicles with the cell
membrane protein SNAP25. SNAP25 can interact with the vesicle protein CSPα in cis-
complexes on the vesicle itself. This sequesters CSPα and prevents the formation of trans-
complexes with SNAP25 on the cell membrane. This trans-interaction, however, promotes
vesicle fusion to the cell membrane. The more often a vesicle has fused to the cell
membrane, the higher its chance is to be contaminated with SNAP25, and the less
competent it is for future rounds of release. The inactivation of ageing synaptic vesicles is
presumably coupled to usage to remove potentially damaged synaptic vesicles from
neurotransmission. This hypothesis is strengthened by the observation of endocytosis
defects and neurite degeneration when aged vesicles are forced to release. I further provide
several timing parameters for key events in the life of synaptic vesicles, which can serve as a
framework towards a quantitative model of the synaptic vesicle life cycle.
SNAP25
Kiss-and-run fusion
Vesicle fusion
Synaptic vesicle recycling
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Synaptotagmin 1
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According to the current theory of synaptic transmission, the amplitude of evoked synaptic potentials correlates with the number of synaptic vesicles released at the presynaptic terminals. Synaptic vesicles in presynaptic boutons constitute two distinct pools, namely, exo/endo cycling and reserve pools (). We defined the vesicles that were endocytosed and exocytosed during high K+ stimulation as the exo/endo cycling vesicle pool. To determine the role of exo/endo cycling vesicle pool in synaptic transmission, we estimated the quantal content electrophysiologically, whereas the pool size was determined optically using fluorescent dye FM1-43. We then manipulated the size of the pool with following treatments. First, to change the state of boutons of nerve terminals, motoneuronal axons were severed. With this treatment, the size of exo/endo cycling vesicle pool decreased together with the quantal content. Second, we promoted the FM1-43 uptake using cyclosporin A, which inhibits calcineurin activities and enhances endocytosis. Cyclosporin A increased the total uptake of FM1-43, but neither the size of exo/endo cycling vesicle pool nor the quantal content changed. Third, we increased the size of exo/endo cycling vesicle pool by forskolin, which enhances synaptic transmission. The forskolin treatment increased both the size of exo/endo cycling vesicle pool and the quantal content. Thus, we found that the quantal content was closely correlated with the size of exo/endo cycling vesicle pool but not necessarily with the total uptake of FM1-43 fluorescence by boutons. The results suggest that vesicles in the exo/endo cycling pool primarily participate in evoked exocytosis of vesicles.
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Developmental Biology
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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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