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Spatial constraints dictate glial territories at murine neuromuscular junctions.

Brill MS, Lichtman JW, Thompson W, Zuo Y, Misgeld T - J. Cell Biol. (2011)

Bottom Line: Adult terminal SCs are arranged in static tile patterns, whereas young SCs dynamically intermingle.The mechanism of developmental glial segregation appears to be spatial competition, in which glial-glial and axonal-glial contacts constrain the territory of single SCs, as shown by four types of experiments: (1) laser ablation of single SCs, which led to immediate territory expansion of neighboring SCs; (2) axon removal by transection, resulting in adult SCs intermingling dynamically; (3) axotomy in mutant mice with blocked axon fragmentation in which intermingling was delayed; and (4) activity blockade, which had no immediate effects.In summary, we conclude that glial cells partition synapses by competing for perisynaptic space.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Integrated Protein Science Munich at the Institute of Neuroscience, Technische Universität München, 80802 Munich, Germany.

ABSTRACT
Schwann cells (SCs), the glial cells of the peripheral nervous system, cover synaptic terminals, allowing them to monitor and modulate neurotransmission. Disruption of glial coverage leads to axon degeneration and synapse loss. The cellular mechanisms that establish and maintain this coverage remain largely unknown. To address this, we labeled single SCs and performed time-lapse imaging experiments. Adult terminal SCs are arranged in static tile patterns, whereas young SCs dynamically intermingle. The mechanism of developmental glial segregation appears to be spatial competition, in which glial-glial and axonal-glial contacts constrain the territory of single SCs, as shown by four types of experiments: (1) laser ablation of single SCs, which led to immediate territory expansion of neighboring SCs; (2) axon removal by transection, resulting in adult SCs intermingling dynamically; (3) axotomy in mutant mice with blocked axon fragmentation in which intermingling was delayed; and (4) activity blockade, which had no immediate effects. In summary, we conclude that glial cells partition synapses by competing for perisynaptic space.

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Ablation of adult terminal SCs in the presence of EtHD. (A) A pseudocolored adult NMJ with three terminal SCs (white, magenta, and cyan). (B) Time-lapse recording of area boxed in A over a period of 5 h showing a two-photon laser–induced ablation of the magenta-colored terminal SC in the presence of EtHD (orange arrowhead indicates the site of EtHD influx). After ablation of the magenta SC, the cyan cell was photobleached (cyan arrowhead). Note the absence of EtHD influx in the bleached terminal SC and the expansion (white arrowheads) of the unbleached (white) terminal SC. (C and D) Confocal view of the same NMJ after fixation and counterstaining with BTX (C) and DAPI (D). The nucleus of the ablated terminal SC is filled with EtHD (orange arrowheads in C and D), whereas the bleached terminal SC is not (cyan arrowheads in C and D). (E) Higher magnification of boxed area in D. The timers shown represent hours/minutes. Bars, 5 µm.
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fig4: Ablation of adult terminal SCs in the presence of EtHD. (A) A pseudocolored adult NMJ with three terminal SCs (white, magenta, and cyan). (B) Time-lapse recording of area boxed in A over a period of 5 h showing a two-photon laser–induced ablation of the magenta-colored terminal SC in the presence of EtHD (orange arrowhead indicates the site of EtHD influx). After ablation of the magenta SC, the cyan cell was photobleached (cyan arrowhead). Note the absence of EtHD influx in the bleached terminal SC and the expansion (white arrowheads) of the unbleached (white) terminal SC. (C and D) Confocal view of the same NMJ after fixation and counterstaining with BTX (C) and DAPI (D). The nucleus of the ablated terminal SC is filled with EtHD (orange arrowheads in C and D), whereas the bleached terminal SC is not (cyan arrowheads in C and D). (E) Higher magnification of boxed area in D. The timers shown represent hours/minutes. Bars, 5 µm.

Mentions: What determines how SCs partition NMJs at different developmental stages? Either the cellular propensity to dynamic exploration could diminish as SCs mature, or, alternatively, the dynamism of adult SCs could be suppressed by external influences. One plausible mechanism would be competition for available perisynaptic space, as SCs consolidate their territory during segregation. To test this hypothesis, we acutely ablated single SCs using a two-photon femtosecond-pulsed laser (Figs. 4 and 5 and Videos 3–6; Galbraith and Terasaki, 2003; Williams et al., 2010). By parking the high-intensity laser beam briefly inside a cell’s nucleus, we could ablate single SCs, as confirmed by ethidium homodimer (EtHD) influx (see Materials and methods; Fig. 4; Reddy et al., 2003). Targeted SCs quickly fragmented, vacating their original territory. Notably, nearby SCs that were bleached by exposure to a conventional continuous-wave laser for single-cell bleaching showed no evidence of phototoxicity (Fig. 4 and Video 3). Within minutes after the demise of the ablated cells, neighboring terminal SCs started to invade the newly vacated territory. Over the course of up to 5 h, the expanding cells engulfed the remnants of the ablated cell and covered the available space (n = 9/10 cases, six triangularis sterni explants; Figs. 4 B, 5 [A–C], and S4 and Video 4). Similarly, when the axonal SC next to an NMJ was ablated, terminal SCs swiftly overgrew the heminode to wrap the denuded axon (n = 5/5 cases, five triangularis sterni explants; Figs. 5 [D–F] and S4 and Video 5). In contrast, axonal SCs that adjoined an NMJ did not invade the synapse after ablation of terminal SCs over the same time period (n = 7/7 cases, four triangularis sterni explants; Figs. 5 [G–I] and S4 and Video 6). Hence, the lack of adult SC dynamism might be a result of spatial competition, in which neighboring cells constantly push against each other without substantial changes in synaptic area they cover. However, axonal SCs seem to be constrained by additional factors at the heminode or by their state of differentiation.


Spatial constraints dictate glial territories at murine neuromuscular junctions.

Brill MS, Lichtman JW, Thompson W, Zuo Y, Misgeld T - J. Cell Biol. (2011)

Ablation of adult terminal SCs in the presence of EtHD. (A) A pseudocolored adult NMJ with three terminal SCs (white, magenta, and cyan). (B) Time-lapse recording of area boxed in A over a period of 5 h showing a two-photon laser–induced ablation of the magenta-colored terminal SC in the presence of EtHD (orange arrowhead indicates the site of EtHD influx). After ablation of the magenta SC, the cyan cell was photobleached (cyan arrowhead). Note the absence of EtHD influx in the bleached terminal SC and the expansion (white arrowheads) of the unbleached (white) terminal SC. (C and D) Confocal view of the same NMJ after fixation and counterstaining with BTX (C) and DAPI (D). The nucleus of the ablated terminal SC is filled with EtHD (orange arrowheads in C and D), whereas the bleached terminal SC is not (cyan arrowheads in C and D). (E) Higher magnification of boxed area in D. The timers shown represent hours/minutes. Bars, 5 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3198169&req=5

fig4: Ablation of adult terminal SCs in the presence of EtHD. (A) A pseudocolored adult NMJ with three terminal SCs (white, magenta, and cyan). (B) Time-lapse recording of area boxed in A over a period of 5 h showing a two-photon laser–induced ablation of the magenta-colored terminal SC in the presence of EtHD (orange arrowhead indicates the site of EtHD influx). After ablation of the magenta SC, the cyan cell was photobleached (cyan arrowhead). Note the absence of EtHD influx in the bleached terminal SC and the expansion (white arrowheads) of the unbleached (white) terminal SC. (C and D) Confocal view of the same NMJ after fixation and counterstaining with BTX (C) and DAPI (D). The nucleus of the ablated terminal SC is filled with EtHD (orange arrowheads in C and D), whereas the bleached terminal SC is not (cyan arrowheads in C and D). (E) Higher magnification of boxed area in D. The timers shown represent hours/minutes. Bars, 5 µm.
Mentions: What determines how SCs partition NMJs at different developmental stages? Either the cellular propensity to dynamic exploration could diminish as SCs mature, or, alternatively, the dynamism of adult SCs could be suppressed by external influences. One plausible mechanism would be competition for available perisynaptic space, as SCs consolidate their territory during segregation. To test this hypothesis, we acutely ablated single SCs using a two-photon femtosecond-pulsed laser (Figs. 4 and 5 and Videos 3–6; Galbraith and Terasaki, 2003; Williams et al., 2010). By parking the high-intensity laser beam briefly inside a cell’s nucleus, we could ablate single SCs, as confirmed by ethidium homodimer (EtHD) influx (see Materials and methods; Fig. 4; Reddy et al., 2003). Targeted SCs quickly fragmented, vacating their original territory. Notably, nearby SCs that were bleached by exposure to a conventional continuous-wave laser for single-cell bleaching showed no evidence of phototoxicity (Fig. 4 and Video 3). Within minutes after the demise of the ablated cells, neighboring terminal SCs started to invade the newly vacated territory. Over the course of up to 5 h, the expanding cells engulfed the remnants of the ablated cell and covered the available space (n = 9/10 cases, six triangularis sterni explants; Figs. 4 B, 5 [A–C], and S4 and Video 4). Similarly, when the axonal SC next to an NMJ was ablated, terminal SCs swiftly overgrew the heminode to wrap the denuded axon (n = 5/5 cases, five triangularis sterni explants; Figs. 5 [D–F] and S4 and Video 5). In contrast, axonal SCs that adjoined an NMJ did not invade the synapse after ablation of terminal SCs over the same time period (n = 7/7 cases, four triangularis sterni explants; Figs. 5 [G–I] and S4 and Video 6). Hence, the lack of adult SC dynamism might be a result of spatial competition, in which neighboring cells constantly push against each other without substantial changes in synaptic area they cover. However, axonal SCs seem to be constrained by additional factors at the heminode or by their state of differentiation.

Bottom Line: Adult terminal SCs are arranged in static tile patterns, whereas young SCs dynamically intermingle.The mechanism of developmental glial segregation appears to be spatial competition, in which glial-glial and axonal-glial contacts constrain the territory of single SCs, as shown by four types of experiments: (1) laser ablation of single SCs, which led to immediate territory expansion of neighboring SCs; (2) axon removal by transection, resulting in adult SCs intermingling dynamically; (3) axotomy in mutant mice with blocked axon fragmentation in which intermingling was delayed; and (4) activity blockade, which had no immediate effects.In summary, we conclude that glial cells partition synapses by competing for perisynaptic space.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Integrated Protein Science Munich at the Institute of Neuroscience, Technische Universität München, 80802 Munich, Germany.

ABSTRACT
Schwann cells (SCs), the glial cells of the peripheral nervous system, cover synaptic terminals, allowing them to monitor and modulate neurotransmission. Disruption of glial coverage leads to axon degeneration and synapse loss. The cellular mechanisms that establish and maintain this coverage remain largely unknown. To address this, we labeled single SCs and performed time-lapse imaging experiments. Adult terminal SCs are arranged in static tile patterns, whereas young SCs dynamically intermingle. The mechanism of developmental glial segregation appears to be spatial competition, in which glial-glial and axonal-glial contacts constrain the territory of single SCs, as shown by four types of experiments: (1) laser ablation of single SCs, which led to immediate territory expansion of neighboring SCs; (2) axon removal by transection, resulting in adult SCs intermingling dynamically; (3) axotomy in mutant mice with blocked axon fragmentation in which intermingling was delayed; and (4) activity blockade, which had no immediate effects. In summary, we conclude that glial cells partition synapses by competing for perisynaptic space.

Show MeSH
Related in: MedlinePlus