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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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Mature segregated terminal SC territories develop from initial glial intermingling. (A and B) Sequential photobleaching of terminal SCs in P11 young (A) and adult (B) nerve–muscle explants from SC-GFP mice. Sequential bleaching steps are depicted in the middle two panels; subtracted and pseudocolored merges of confocal images are shown in the rightmost panels. (C) Terminal SC area overlap as a percentage of total synaptic (BTX positive) area (young: 11.0 ± 1.5%, n = 30 SC pairs, 10 triangularis sterni muscles; adult: 1.7 ± 0.4%, n = 26 SC pairs, 9 triangularis sterni muscles; *, P < 0.01 using a t test; data are represented as the mean of SC pairs + SEM). (D and E) Terminal SC segregation at young versus mature ages. (D) Three examples are shown: an extensively intermingled young SC pair (8.5% minimum), an intermediate example (36.5%), and a mature highly segregated terminal SC pair (53.7%; maximum segregation index measured). Color-coded dots indicate centroids of individual SCs, and lines indicate the length of the NMJ at the axis through the centroids. (E) Quantification of age-dependent segregation (calculated as the distance of the centroids over the length of the NMJ): young (P7–11; 30.4 ± 1.6%, n = 24 SC pairs, six triangularis sterni muscles) versus adult (44.6 ± 1.3%, n = 23 SC pairs, three triangularis sterni muscles; mean of SC pairs + SEM; *, P < 0.01 using a t test). Bars, 5 µm.
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fig1: Mature segregated terminal SC territories develop from initial glial intermingling. (A and B) Sequential photobleaching of terminal SCs in P11 young (A) and adult (B) nerve–muscle explants from SC-GFP mice. Sequential bleaching steps are depicted in the middle two panels; subtracted and pseudocolored merges of confocal images are shown in the rightmost panels. (C) Terminal SC area overlap as a percentage of total synaptic (BTX positive) area (young: 11.0 ± 1.5%, n = 30 SC pairs, 10 triangularis sterni muscles; adult: 1.7 ± 0.4%, n = 26 SC pairs, 9 triangularis sterni muscles; *, P < 0.01 using a t test; data are represented as the mean of SC pairs + SEM). (D and E) Terminal SC segregation at young versus mature ages. (D) Three examples are shown: an extensively intermingled young SC pair (8.5% minimum), an intermediate example (36.5%), and a mature highly segregated terminal SC pair (53.7%; maximum segregation index measured). Color-coded dots indicate centroids of individual SCs, and lines indicate the length of the NMJ at the axis through the centroids. (E) Quantification of age-dependent segregation (calculated as the distance of the centroids over the length of the NMJ): young (P7–11; 30.4 ± 1.6%, n = 24 SC pairs, six triangularis sterni muscles) versus adult (44.6 ± 1.3%, n = 23 SC pairs, three triangularis sterni muscles; mean of SC pairs + SEM; *, P < 0.01 using a t test). Bars, 5 µm.

Mentions: Because of direct apposition between terminal SCs, no clear borders between individual cells could be defined in NMJs of SC-GFP mice by conventional confocal microscopy alone (Figs. 1 [A and B] and S1). To delineate individual SC territories, we developed two independent techniques (for details, see Materials and methods): we either sequentially dye filled individual SCs with rhodamine dextran (Fig. S1 A) or used sequential photobleaching (Fig. 1, A and B). Three types of SC contacts became apparent (Fig. S1): contacts that involved axonal SCs (axonal–axonal and axonal–terminal) and contacts between terminal SCs (terminal–terminal). Axonal SCs were delineated by gaps indicative of nodal structures, which were surrounded by immunoreactivity for the paranodal marker contactin associated protein-1 (Caspr; Fig. S1 B; Scherer, 1996). A heminode, with unilateral Caspr immunoreactivity, defined the synaptic entry point and was never crossed by glial processes (Fig. S1 B; n > 70 terminal SCs and 7 axonal SCs that participated in a heminode). Within the synapse (where Caspr accumulations are absent), single-cell labeling also revealed clearly defined glial territories that tile the mature NMJ (Figs. 1 B and S1 [A and B]). Mature terminal SCs possess multiple processes (sternomastoid: 4.9 ± 0.4 per cell, n = 32 SCs, 12 muscles; triangularis sterni: 4.4 ± 0.2, n = 31 SCs, 7 muscles) that show almost no overlap at SC–SC contact sites (Fig.1 C). Despite these sharp boundaries, extensive cell–cell contacts and, hence, potential communication sites between neighboring SCs exist. When we coinjected a small molecular tag (neurobiotin; ∼300 D; Kristan et al., 2000) together with rhodamine dextran, we found that terminal SC pairs were coupled at 59% of NMJs (n = 10/17 NMJs, 17 muscles; Fig. S1 C), compatible with the presence of gap junctions. In contrast, coupling between terminal and axonal SCs was never observed (n = 0/9 NMJs, nine muscles). Thus, in undisturbed adult NMJs, terminal and axonal SCs belong to distinct compartments. Individual terminal SCs cover distinct synaptic territories but at the same time can form a functional syncytium, which might allow for intercellular signaling.


Spatial constraints dictate glial territories at murine neuromuscular junctions.

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

Mature segregated terminal SC territories develop from initial glial intermingling. (A and B) Sequential photobleaching of terminal SCs in P11 young (A) and adult (B) nerve–muscle explants from SC-GFP mice. Sequential bleaching steps are depicted in the middle two panels; subtracted and pseudocolored merges of confocal images are shown in the rightmost panels. (C) Terminal SC area overlap as a percentage of total synaptic (BTX positive) area (young: 11.0 ± 1.5%, n = 30 SC pairs, 10 triangularis sterni muscles; adult: 1.7 ± 0.4%, n = 26 SC pairs, 9 triangularis sterni muscles; *, P < 0.01 using a t test; data are represented as the mean of SC pairs + SEM). (D and E) Terminal SC segregation at young versus mature ages. (D) Three examples are shown: an extensively intermingled young SC pair (8.5% minimum), an intermediate example (36.5%), and a mature highly segregated terminal SC pair (53.7%; maximum segregation index measured). Color-coded dots indicate centroids of individual SCs, and lines indicate the length of the NMJ at the axis through the centroids. (E) Quantification of age-dependent segregation (calculated as the distance of the centroids over the length of the NMJ): young (P7–11; 30.4 ± 1.6%, n = 24 SC pairs, six triangularis sterni muscles) versus adult (44.6 ± 1.3%, n = 23 SC pairs, three triangularis sterni muscles; mean of SC pairs + SEM; *, P < 0.01 using a t test). Bars, 5 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig1: Mature segregated terminal SC territories develop from initial glial intermingling. (A and B) Sequential photobleaching of terminal SCs in P11 young (A) and adult (B) nerve–muscle explants from SC-GFP mice. Sequential bleaching steps are depicted in the middle two panels; subtracted and pseudocolored merges of confocal images are shown in the rightmost panels. (C) Terminal SC area overlap as a percentage of total synaptic (BTX positive) area (young: 11.0 ± 1.5%, n = 30 SC pairs, 10 triangularis sterni muscles; adult: 1.7 ± 0.4%, n = 26 SC pairs, 9 triangularis sterni muscles; *, P < 0.01 using a t test; data are represented as the mean of SC pairs + SEM). (D and E) Terminal SC segregation at young versus mature ages. (D) Three examples are shown: an extensively intermingled young SC pair (8.5% minimum), an intermediate example (36.5%), and a mature highly segregated terminal SC pair (53.7%; maximum segregation index measured). Color-coded dots indicate centroids of individual SCs, and lines indicate the length of the NMJ at the axis through the centroids. (E) Quantification of age-dependent segregation (calculated as the distance of the centroids over the length of the NMJ): young (P7–11; 30.4 ± 1.6%, n = 24 SC pairs, six triangularis sterni muscles) versus adult (44.6 ± 1.3%, n = 23 SC pairs, three triangularis sterni muscles; mean of SC pairs + SEM; *, P < 0.01 using a t test). Bars, 5 µm.
Mentions: Because of direct apposition between terminal SCs, no clear borders between individual cells could be defined in NMJs of SC-GFP mice by conventional confocal microscopy alone (Figs. 1 [A and B] and S1). To delineate individual SC territories, we developed two independent techniques (for details, see Materials and methods): we either sequentially dye filled individual SCs with rhodamine dextran (Fig. S1 A) or used sequential photobleaching (Fig. 1, A and B). Three types of SC contacts became apparent (Fig. S1): contacts that involved axonal SCs (axonal–axonal and axonal–terminal) and contacts between terminal SCs (terminal–terminal). Axonal SCs were delineated by gaps indicative of nodal structures, which were surrounded by immunoreactivity for the paranodal marker contactin associated protein-1 (Caspr; Fig. S1 B; Scherer, 1996). A heminode, with unilateral Caspr immunoreactivity, defined the synaptic entry point and was never crossed by glial processes (Fig. S1 B; n > 70 terminal SCs and 7 axonal SCs that participated in a heminode). Within the synapse (where Caspr accumulations are absent), single-cell labeling also revealed clearly defined glial territories that tile the mature NMJ (Figs. 1 B and S1 [A and B]). Mature terminal SCs possess multiple processes (sternomastoid: 4.9 ± 0.4 per cell, n = 32 SCs, 12 muscles; triangularis sterni: 4.4 ± 0.2, n = 31 SCs, 7 muscles) that show almost no overlap at SC–SC contact sites (Fig.1 C). Despite these sharp boundaries, extensive cell–cell contacts and, hence, potential communication sites between neighboring SCs exist. When we coinjected a small molecular tag (neurobiotin; ∼300 D; Kristan et al., 2000) together with rhodamine dextran, we found that terminal SC pairs were coupled at 59% of NMJs (n = 10/17 NMJs, 17 muscles; Fig. S1 C), compatible with the presence of gap junctions. In contrast, coupling between terminal and axonal SCs was never observed (n = 0/9 NMJs, nine muscles). Thus, in undisturbed adult NMJs, terminal and axonal SCs belong to distinct compartments. Individual terminal SCs cover distinct synaptic territories but at the same time can form a functional syncytium, which might allow for intercellular signaling.

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