| 1. |
- Granseth, Björn, et al.
(författare)
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Clathrin-mediated endocytosis : the physiological mechanism of vesicle retrieval at hippocampal synapses
- 2007
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Ingår i: Journal of Physiology. - : Wiley. - 0022-3751 .- 1469-7793. ; 585:3, s. 681-686
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Forskningsöversikt (refereegranskat)abstract
- The maintenance of synaptic transmission requires that vesicles are recycled after releasing neurotransmitter. Several modes of retrieval have been proposed to operate at small synaptic terminals of central neurons, but the relative importance of these has been controversial. It is established that synaptic vesicles can collapse on fusion and the machinery for retrieving this membrane by clathrin-mediated endocytosis (CME) is enriched in the presynaptic terminal. But it has also been suggested that the majority of vesicles released by physiological stimulation are recycled by a second, faster mechanism called 'kiss-and-run', which operates in 1 s or less to retrieve a vesicle before it has collapsed. The most recent evidence argues against the occurrence of 'kiss-and-run' in hippocampal synapses. First, an improved fluorescent reporter of exocytosis (sypHy), indicates that only a slow mode of endocytosis (tau = 15 s) operates when vesicle fusion is triggered by a single nerve impulse or short burst. Second, this retrieval mechanism is blocked by overexpressing the C-terminal fragment of AP180 or by knockdown of clathrin using RNAi. Third, vesicle fusion is associated with the movement of clathrin and vesicle proteins out of the synapse into the neighbouring axon. These observations indicate that clathrin-mediated endocytosis is the major, if not exclusive, mechanism of retrieval in small hippocampal synapses.
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| 2. |
- Granseth, Björn, et al.
(författare)
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Clathrin-mediated endocytosis is the dominant mechanism of vesicle retrieval at hippocampal synapses
- 2006
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Ingår i: Neuron. - : Elsevier BV. - 0896-6273 .- 1097-4199. ; 51:6, s. 773-786
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Tidskriftsartikel (refereegranskat)abstract
- The maintenance of synaptic transmission requires that vesicles be recycled after releasing neurotransmitter. Several modes of retrieval have been proposed to operate at small synaptic terminals of central neurons, including a fast "kiss-and-run" mechanism that releases neurotransmitter through a fusion pore. Using an improved fluorescent reporter comprising pHluorin fused to synaptophysin, we find that only a slow mode of endocytosis (tau = 15 s) operates at hippocampal synapses when vesicle fusion is triggered by a single nerve impulse or short burst. This retrieval mechanism is blocked by overexpression of the C-terminal fragment of AP180 or by knockdown of clathrin using RNAi, and it is associated with the movement of clathrin and vesicle proteins out of the synapse. These results indicate that clathrin-mediated endocytosis is the major, if not exclusive, mechanism of vesicle retrieval after physiological stimuli.
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| 3. |
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| 4. |
- Granseth, Björn, 1973-, et al.
(författare)
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The role of endocytosis in regulating the strength of hippocampal synapses
- 2008
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Ingår i: Journal of Physiology. - : Wiley. - 0022-3751 .- 1469-7793. ; 586, s. 5969-5982
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Tidskriftsartikel (refereegranskat)abstract
- The readily releasable pool of vesicles (RRP) varies in size during synaptic activity and is replenished by recruitment from the reserve pool as well as vesicle retrieval after fusion. To investigate which of these steps is rate limiting in supplying vesicles to the RRP, we measured the effects of changes in temperature in cultured hippocampal neurons, where higher average rates of release can be maintained as the temperature is increased. Using a pHluorin-based reporter of exocytosis and endocytosis (sypHy), we find that changes in temperature between 25 degrees C and 35 degrees C do not significantly alter the rate of recruitment from the reserve pool. In contrast, the time constant of endocytosis fell from approximately 17 s at 25 degrees C to approximately 10 s at 35 degrees C (Q(10) = 1.7), while the time constant of vesicle reacidification fell from approximately 5.5 s to approximately 1 s (Q(10) = 5.5). A kinetic model of the vesicle cycle constructed using measured parameters was found to describe variations in vesicle release rate observed during long trains of spikes as well as recovery from synaptic depression after bursts of activity. These results indicate that endocytosis operating with time constants of 10-15 s is the rate-limiting process determining replenishment of the RRP during long-term activity. A fast mode of vesicle retrieval could not be detected at any temperature, nor was it necessary to invoke such a mechanism to account for use-dependent changes in synaptic release probability.
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| 5. |
- Royle, Stephen J, et al.
(författare)
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Imaging phluorin-based probes at hippocampal synapses
- 2008
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Ingår i: Membrane Trafficking. - Totowa, NJ : Humana Press. - 9781597452618 ; , s. 293-303
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- Accurate measurement of synaptic vesicle exocytosis and endocytosis is crucial to understanding the molecular basis of synaptic transmission. The fusion of a pH-sensitive green fluorescent protein (pHluorin) to various synaptic vesicle proteins has allowed the study of synaptic vesicle recycling in real time. Two such probes, synaptopHluorin and sypHy, have been imaged at synapses of hippocampal neurons in culture. The combination of these reporters with techniques for molecular interference, such as RNAi allows for the study of molecules involved in synaptic vesicle recycling. Here the authors describe methods for the culture and transfection of hippocampal neurons, imaging of pHluorin-based probes at synapses and analysis of pHluorin signals down to the resolution of individual synaptic vesicles.
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