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A vesicle superpool spans multiple presynaptic terminals in hippocampal neurons.

Staras K, Branco T, Burden JJ, Pozo K, Darcy K, Marra V, Ratnayaka A, Goda Y - Neuron (2010)

Bottom Line: Here, we demonstrate a vesicle pool that is not confined to a synapse but spans multiple terminals.In acute hippocampal slices we show that the mobile vesicle pool is also a feature of native brain tissue.We also demonstrate that superpool vesicles are available to synapses during stimulation, providing an extension of the classical recycling pool.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, University of Sussex, Brighton, UK. k.staras@sussex.ac.uk

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Related in: MedlinePlus

Ultrastructural Readout of Functional Vesicle Sharing from a Target Synapse(A) Experimental scheme. (B) Ultrastructural reconstruction of target process showing axon, dendrite, and SV clusters (red). (C) Sample EM images from synapses in (B), fixed after ∼5 min. Top left (“s”): unbleached source synapse. Recycling vesicles (PC+) have dark lumen (arrowheads) and nonrecycling vesicles (PC−) have clear lumen, which are readily distinguishable (inset). (D) Reconstruction of vesicle clusters from “source” synapse and synapse “2” from (B). Green, active zone. (E) Full reconstruction of axon and vesicles from (B). (F) A second example illustrating lateral spread of recycling vesicles arising from the synaptic source into bleached synapses. (G) Summary of vesicle sharing from a source terminal to synaptic neighbors showing PC+ vesicles as a percentage of total vesicle count for each synapse (1, 2, 3) adjacent to an unbleached source synapse, 0 (blue) or 5 min (red) after photobleaching. Intersynaptic distances were not significantly different for control (5.4 ± 0.2 μm) versus experimental conditions (5.5 ± 0.5 μm) (t test, p > 0.92). Values are mean ± SEM. (H) Sample EM images of synapses from 0 min control group: unbleached synapse (top) and adjacent photobleached synapse (bottom).
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fig2: Ultrastructural Readout of Functional Vesicle Sharing from a Target Synapse(A) Experimental scheme. (B) Ultrastructural reconstruction of target process showing axon, dendrite, and SV clusters (red). (C) Sample EM images from synapses in (B), fixed after ∼5 min. Top left (“s”): unbleached source synapse. Recycling vesicles (PC+) have dark lumen (arrowheads) and nonrecycling vesicles (PC−) have clear lumen, which are readily distinguishable (inset). (D) Reconstruction of vesicle clusters from “source” synapse and synapse “2” from (B). Green, active zone. (E) Full reconstruction of axon and vesicles from (B). (F) A second example illustrating lateral spread of recycling vesicles arising from the synaptic source into bleached synapses. (G) Summary of vesicle sharing from a source terminal to synaptic neighbors showing PC+ vesicles as a percentage of total vesicle count for each synapse (1, 2, 3) adjacent to an unbleached source synapse, 0 (blue) or 5 min (red) after photobleaching. Intersynaptic distances were not significantly different for control (5.4 ± 0.2 μm) versus experimental conditions (5.5 ± 0.5 μm) (t test, p > 0.92). Values are mean ± SEM. (H) Sample EM images of synapses from 0 min control group: unbleached synapse (top) and adjacent photobleached synapse (bottom).

Mentions: SypI-Dendra2 provides an informative readout of vesicle sharing dynamics but offers a restricted view of the detailed organization of the shared vesicle pool. For example, conventional light microscopy limits the visualization of mobile vesicle traffic to large and clustered vesicle packets. It is not clear whether such vesicle modules reflect the true organization of the shared vesicle pool or if single vesicles could also be mobilized between boutons. Also, SypI-Dendra2 does not discriminate between functionally active vesicles and those in the nonrecycling pool, even though the vesicle dynamics may be dependent on the functional class of vesicles or their recent history. To address these issues directly, we employed a correlative fluorescence and EM method to examine properties of the shared pool in ultrastructural detail (Darcy et al., 2006a, 2006b). The total recycling pool in synaptic terminals was labeled with a fixable form of FM1-43 dye (Betz and Bewick, 1992; Ryan et al., 1993). Single axonal processes with multiple sequential FM-dye-labeled synapses were identified and subjected to a reverse FRAP protocol (Figure 2A) in which fluorescence of a single target synapse was preserved while flanking terminals were rapidly photobleached. Neurons were fixed after 5 min, FM-dye was photoconverted (Darcy et al., 2006a; Harata et al., 2001; Rizzoli and Betz, 2004; Schikorski and Stevens, 2001), and samples were processed for serial section EM. In this way, recycling vesicles contributed by a single target bouton to the neighboring regions over 5 min could be visualized and quantified. As controls, target terminals were photobleached and fixed immediately.


A vesicle superpool spans multiple presynaptic terminals in hippocampal neurons.

Staras K, Branco T, Burden JJ, Pozo K, Darcy K, Marra V, Ratnayaka A, Goda Y - Neuron (2010)

Ultrastructural Readout of Functional Vesicle Sharing from a Target Synapse(A) Experimental scheme. (B) Ultrastructural reconstruction of target process showing axon, dendrite, and SV clusters (red). (C) Sample EM images from synapses in (B), fixed after ∼5 min. Top left (“s”): unbleached source synapse. Recycling vesicles (PC+) have dark lumen (arrowheads) and nonrecycling vesicles (PC−) have clear lumen, which are readily distinguishable (inset). (D) Reconstruction of vesicle clusters from “source” synapse and synapse “2” from (B). Green, active zone. (E) Full reconstruction of axon and vesicles from (B). (F) A second example illustrating lateral spread of recycling vesicles arising from the synaptic source into bleached synapses. (G) Summary of vesicle sharing from a source terminal to synaptic neighbors showing PC+ vesicles as a percentage of total vesicle count for each synapse (1, 2, 3) adjacent to an unbleached source synapse, 0 (blue) or 5 min (red) after photobleaching. Intersynaptic distances were not significantly different for control (5.4 ± 0.2 μm) versus experimental conditions (5.5 ± 0.5 μm) (t test, p > 0.92). Values are mean ± SEM. (H) Sample EM images of synapses from 0 min control group: unbleached synapse (top) and adjacent photobleached synapse (bottom).
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Related In: Results  -  Collection

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Show All Figures
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fig2: Ultrastructural Readout of Functional Vesicle Sharing from a Target Synapse(A) Experimental scheme. (B) Ultrastructural reconstruction of target process showing axon, dendrite, and SV clusters (red). (C) Sample EM images from synapses in (B), fixed after ∼5 min. Top left (“s”): unbleached source synapse. Recycling vesicles (PC+) have dark lumen (arrowheads) and nonrecycling vesicles (PC−) have clear lumen, which are readily distinguishable (inset). (D) Reconstruction of vesicle clusters from “source” synapse and synapse “2” from (B). Green, active zone. (E) Full reconstruction of axon and vesicles from (B). (F) A second example illustrating lateral spread of recycling vesicles arising from the synaptic source into bleached synapses. (G) Summary of vesicle sharing from a source terminal to synaptic neighbors showing PC+ vesicles as a percentage of total vesicle count for each synapse (1, 2, 3) adjacent to an unbleached source synapse, 0 (blue) or 5 min (red) after photobleaching. Intersynaptic distances were not significantly different for control (5.4 ± 0.2 μm) versus experimental conditions (5.5 ± 0.5 μm) (t test, p > 0.92). Values are mean ± SEM. (H) Sample EM images of synapses from 0 min control group: unbleached synapse (top) and adjacent photobleached synapse (bottom).
Mentions: SypI-Dendra2 provides an informative readout of vesicle sharing dynamics but offers a restricted view of the detailed organization of the shared vesicle pool. For example, conventional light microscopy limits the visualization of mobile vesicle traffic to large and clustered vesicle packets. It is not clear whether such vesicle modules reflect the true organization of the shared vesicle pool or if single vesicles could also be mobilized between boutons. Also, SypI-Dendra2 does not discriminate between functionally active vesicles and those in the nonrecycling pool, even though the vesicle dynamics may be dependent on the functional class of vesicles or their recent history. To address these issues directly, we employed a correlative fluorescence and EM method to examine properties of the shared pool in ultrastructural detail (Darcy et al., 2006a, 2006b). The total recycling pool in synaptic terminals was labeled with a fixable form of FM1-43 dye (Betz and Bewick, 1992; Ryan et al., 1993). Single axonal processes with multiple sequential FM-dye-labeled synapses were identified and subjected to a reverse FRAP protocol (Figure 2A) in which fluorescence of a single target synapse was preserved while flanking terminals were rapidly photobleached. Neurons were fixed after 5 min, FM-dye was photoconverted (Darcy et al., 2006a; Harata et al., 2001; Rizzoli and Betz, 2004; Schikorski and Stevens, 2001), and samples were processed for serial section EM. In this way, recycling vesicles contributed by a single target bouton to the neighboring regions over 5 min could be visualized and quantified. As controls, target terminals were photobleached and fixed immediately.

Bottom Line: Here, we demonstrate a vesicle pool that is not confined to a synapse but spans multiple terminals.In acute hippocampal slices we show that the mobile vesicle pool is also a feature of native brain tissue.We also demonstrate that superpool vesicles are available to synapses during stimulation, providing an extension of the classical recycling pool.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, University of Sussex, Brighton, UK. k.staras@sussex.ac.uk

Show MeSH
Related in: MedlinePlus