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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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SC segregation is axon dependent. (A–D) Single-cell labeling of an NMJ 31 h after transection of the intercostal nerve. (A and B) Terminal SCs overlap but remain restricted within the synaptic gutter (labeled in B with BTX). (C and D) Time-lapse recording spanning 1 h demonstrates rapid growth (C) and retraction (D) of terminal SC processes within the synaptic gutter (depicted areas boxed in A). (E–G) 5 d after nerve transcection, terminal SCs have started to sprout beyond the synapse. (F and G) Terminal SCs exhibit extrasynaptic growth cone–like structures, which grow (F) and collapse (G) rapidly over a period of 1–2 h (depicted areas boxed in E). The timers shown represent hours/minutes. Bars, 5 µm.
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fig6: SC segregation is axon dependent. (A–D) Single-cell labeling of an NMJ 31 h after transection of the intercostal nerve. (A and B) Terminal SCs overlap but remain restricted within the synaptic gutter (labeled in B with BTX). (C and D) Time-lapse recording spanning 1 h demonstrates rapid growth (C) and retraction (D) of terminal SC processes within the synaptic gutter (depicted areas boxed in A). (E–G) 5 d after nerve transcection, terminal SCs have started to sprout beyond the synapse. (F and G) Terminal SCs exhibit extrasynaptic growth cone–like structures, which grow (F) and collapse (G) rapidly over a period of 1–2 h (depicted areas boxed in E). The timers shown represent hours/minutes. Bars, 5 µm.

Mentions: Although SCs laterally abut other SCs, they are also in contact with the underlying axon terminal (Hall and Sanes, 1993). Hence, SCs could be similarly space restrained by axons, predicting that removal of axons would allow for SC dynamism and intermingling. Indeed, previous studies have demonstrated that in chronic stages after axotomy, SCs form long processes outside of NMJs, which serve as bridges to guide regeneration (Reynolds and Woolf, 1992; Kang et al., 2003). To explore whether such remodeling is accompanied by glial intermingling and dynamism, we performed single-cell labeling experiments at various times after transection of motor axons. Transection of motor nerves leads to axonal fragmentation of axons by Wallerian degeneration after a lag phase of ∼12–14 h (Coleman and Freeman, 2010). SCs remained unchanged during the lag period, suggesting that solely transecting the axon was not sufficient to induce glial dynamism. The picture changed as soon as fragmentation set in. At this point, SCs engulfed and digested axonal fragments (Fig. S5). 1 d after axotomy (>24 h), single-cell labeling using photobleaching revealed evidence of intrasynaptic growth. For example, Fig. 6 (A–D) shows an NMJ 1 d after axotomy, which looks normal when all cells are labeled but reveals glial intermingling after single-cell labeling. Early formed processes stayed exclusively within the synaptic gutter or grew toward the axonal SC at the former axonal entry point (Fig. 6 [C and D] and Video 7). Quantification confirmed that SCs on denervated NMJs screened significantly more territory compared with control adult SCs (Fig. S4). At later time points (>48 h), SC growth and intermingling continued, so that the length overlap between terminal SC processes increased from a baseline of 9.0 ± 2 to 55.7 ± 4% (n = 38 pairs of SC processes each; P < 0.001 using a t test). Concomitantly, terminal SCs exhibit long processes inside and outside of the denervated synapse, sometimes with growth cone–like tips that rapidly extended and collapsed (Fig. 6, E–G). The SC boundary between the preterminal axon and the synapse proper was disrupted, with axonal SCs projecting processes into NMJs (n = 7/7 axonal SC) and terminal SCs projecting back into the SC tube (n = 29/78 terminal SCs). After reinnervation, segregated SC territories are reestablished, as 1 mo after denervation, SC processes recovered normal length (control: 13.9 ± 0.8 µm, n = 27 SCs; denervation: 27.3 ± 2.1 µm, n = 28 SCs; reinnervation: 12.3 ± 0.6 µm, n = 29 SCs) and overlap (reinnervation: 8.8 ± 2%, n = 38 pairs of SC processes; P > 0.9 between control and reinnervation using a t test).


Spatial constraints dictate glial territories at murine neuromuscular junctions.

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

SC segregation is axon dependent. (A–D) Single-cell labeling of an NMJ 31 h after transection of the intercostal nerve. (A and B) Terminal SCs overlap but remain restricted within the synaptic gutter (labeled in B with BTX). (C and D) Time-lapse recording spanning 1 h demonstrates rapid growth (C) and retraction (D) of terminal SC processes within the synaptic gutter (depicted areas boxed in A). (E–G) 5 d after nerve transcection, terminal SCs have started to sprout beyond the synapse. (F and G) Terminal SCs exhibit extrasynaptic growth cone–like structures, which grow (F) and collapse (G) rapidly over a period of 1–2 h (depicted areas boxed in E). The timers shown represent hours/minutes. Bars, 5 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3198169&req=5

fig6: SC segregation is axon dependent. (A–D) Single-cell labeling of an NMJ 31 h after transection of the intercostal nerve. (A and B) Terminal SCs overlap but remain restricted within the synaptic gutter (labeled in B with BTX). (C and D) Time-lapse recording spanning 1 h demonstrates rapid growth (C) and retraction (D) of terminal SC processes within the synaptic gutter (depicted areas boxed in A). (E–G) 5 d after nerve transcection, terminal SCs have started to sprout beyond the synapse. (F and G) Terminal SCs exhibit extrasynaptic growth cone–like structures, which grow (F) and collapse (G) rapidly over a period of 1–2 h (depicted areas boxed in E). The timers shown represent hours/minutes. Bars, 5 µm.
Mentions: Although SCs laterally abut other SCs, they are also in contact with the underlying axon terminal (Hall and Sanes, 1993). Hence, SCs could be similarly space restrained by axons, predicting that removal of axons would allow for SC dynamism and intermingling. Indeed, previous studies have demonstrated that in chronic stages after axotomy, SCs form long processes outside of NMJs, which serve as bridges to guide regeneration (Reynolds and Woolf, 1992; Kang et al., 2003). To explore whether such remodeling is accompanied by glial intermingling and dynamism, we performed single-cell labeling experiments at various times after transection of motor axons. Transection of motor nerves leads to axonal fragmentation of axons by Wallerian degeneration after a lag phase of ∼12–14 h (Coleman and Freeman, 2010). SCs remained unchanged during the lag period, suggesting that solely transecting the axon was not sufficient to induce glial dynamism. The picture changed as soon as fragmentation set in. At this point, SCs engulfed and digested axonal fragments (Fig. S5). 1 d after axotomy (>24 h), single-cell labeling using photobleaching revealed evidence of intrasynaptic growth. For example, Fig. 6 (A–D) shows an NMJ 1 d after axotomy, which looks normal when all cells are labeled but reveals glial intermingling after single-cell labeling. Early formed processes stayed exclusively within the synaptic gutter or grew toward the axonal SC at the former axonal entry point (Fig. 6 [C and D] and Video 7). Quantification confirmed that SCs on denervated NMJs screened significantly more territory compared with control adult SCs (Fig. S4). At later time points (>48 h), SC growth and intermingling continued, so that the length overlap between terminal SC processes increased from a baseline of 9.0 ± 2 to 55.7 ± 4% (n = 38 pairs of SC processes each; P < 0.001 using a t test). Concomitantly, terminal SCs exhibit long processes inside and outside of the denervated synapse, sometimes with growth cone–like tips that rapidly extended and collapsed (Fig. 6, E–G). The SC boundary between the preterminal axon and the synapse proper was disrupted, with axonal SCs projecting processes into NMJs (n = 7/7 axonal SC) and terminal SCs projecting back into the SC tube (n = 29/78 terminal SCs). After reinnervation, segregated SC territories are reestablished, as 1 mo after denervation, SC processes recovered normal length (control: 13.9 ± 0.8 µm, n = 27 SCs; denervation: 27.3 ± 2.1 µm, n = 28 SCs; reinnervation: 12.3 ± 0.6 µm, n = 29 SCs) and overlap (reinnervation: 8.8 ± 2%, n = 38 pairs of SC processes; P > 0.9 between control and reinnervation using a t test).

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