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Reversing the outcome of synapse elimination at developing neuromuscular junctions in vivo: evidence for synaptic competition and its mechanism.

Turney SG, Lichtman JW - PLoS Biol. (2012)

Bottom Line: Indeed, during normal development we observed withdrawal followed by takeover.The stimulus for axon growth is not postsynaptic cell inactivity because axons grow into unoccupied sites even when target cells are functionally innervated.These results demonstrate competition at the synaptic level and enable us to provide a conceptual framework for understanding this form of synaptic plasticity.

View Article: PubMed Central - PubMed

Affiliation: Center for Brain Science and Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, United States of America. sturney@mcb.harvard.edu

ABSTRACT
During mammalian development, neuromuscular junctions and some other postsynaptic cells transition from multiple- to single-innervation as synaptic sites are exchanged between different axons. It is unclear whether one axon invades synaptic sites to drive off other inputs or alternatively axons expand their territory in response to sites vacated by other axons. Here we show that soon-to-be-eliminated axons rapidly reverse fate and grow to occupy vacant sites at a neuromuscular junction after laser removal of a stronger input. This reversal supports the idea that axons take over sites that were previously vacated. Indeed, during normal development we observed withdrawal followed by takeover. The stimulus for axon growth is not postsynaptic cell inactivity because axons grow into unoccupied sites even when target cells are functionally innervated. These results demonstrate competition at the synaptic level and enable us to provide a conceptual framework for understanding this form of synaptic plasticity.

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Time course of removal of laser-target input and takeover by another at developing neuromuscular junction.(A) In vivo image of region in a P7 mouse sternomastoid muscle. Boxed area shows a neuromuscular junction innervated by two axons (green, YFP-filled axons; red, α-bungarotoxin-tagged acetylcholine receptors). The axon on the right in the boxed region was irradiated with a mode-locked infrared laser at the site of the circle. (B) The same region of muscle reimaged 27 h later showing that the irradiated axon has completely disappeared. (C) Time-lapse imaging reveals that the remaining axon grows to occupy the sites that were vacated by the damaged axon (arrow points to site of irradiation). Within 0.5 h of irradiation the right axon's terminal branches swell and clearly reveal that the undamaged axon, that remained brightly fluorescent, terminated in a bulb. The swelling of the damaged axon deformed the shape of the intact axon's terminal bulb (panels at 0.8, 1, and 1.5 h). At 1.8 h, the bulb recovered its original shape, presumably because the damaged input had lost its turgor presumably due to membrane leakage. Over the next hour, the fluorescence in the damaged terminals became fainter, its axon fragmented (see 1.8 h), and it largely became invisible. Reimaging the junction 27 h after damage showed that the remaining axon branched to occupy many of the sites previously occupied by the laser irradiated axon. Scale bar, 20 µm.
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pbio-1001352-g001: Time course of removal of laser-target input and takeover by another at developing neuromuscular junction.(A) In vivo image of region in a P7 mouse sternomastoid muscle. Boxed area shows a neuromuscular junction innervated by two axons (green, YFP-filled axons; red, α-bungarotoxin-tagged acetylcholine receptors). The axon on the right in the boxed region was irradiated with a mode-locked infrared laser at the site of the circle. (B) The same region of muscle reimaged 27 h later showing that the irradiated axon has completely disappeared. (C) Time-lapse imaging reveals that the remaining axon grows to occupy the sites that were vacated by the damaged axon (arrow points to site of irradiation). Within 0.5 h of irradiation the right axon's terminal branches swell and clearly reveal that the undamaged axon, that remained brightly fluorescent, terminated in a bulb. The swelling of the damaged axon deformed the shape of the intact axon's terminal bulb (panels at 0.8, 1, and 1.5 h). At 1.8 h, the bulb recovered its original shape, presumably because the damaged input had lost its turgor presumably due to membrane leakage. Over the next hour, the fluorescence in the damaged terminals became fainter, its axon fragmented (see 1.8 h), and it largely became invisible. Reimaging the junction 27 h after damage showed that the remaining axon branched to occupy many of the sites previously occupied by the laser irradiated axon. Scale bar, 20 µm.

Mentions: Damage to axons typically evolved over 30–45 min and the whole process of axon removal required many hours. Even though we observed bleaching of the axon segment at the time of irradiation, evidence for structural damage only became apparent within 10–20 min (see Figures 1–3). Signs of axon damage included dramatic swelling of the axon distal to the site of laser focus and a progressive widening of the region of non-fluorescence both distal and proximal to the laser irradiation site. Presumably this loss of fluorescence is secondary to leakage of proteins from the cytoplasm at the damage site. This phase which typically lasted up to several hours was followed by the complete disappearance of the distal axon save for occasionally a few small disconnected fluorescent fragments that ultimately all disappeared by 10 h. In the proximal direction the damage initiated a die-back that was reminiscent both in time course and scale of “acute axonal degeneration” of damaged central axons [26]. Typically, the die-back stopped at the proximal branch point (Figure 2), although sometimes it extended anterogradely from the branch point to cause the disappearance of other terminal branches. If the fluorescence at the laser spot recovered after several minutes, that was an indication that the fluorescence in the axonal branch had been bleached but the axon was not seriously damaged because no subsequent changes were noted over the next half hour to hour, or the following day (see Materials and Methods for details).


Reversing the outcome of synapse elimination at developing neuromuscular junctions in vivo: evidence for synaptic competition and its mechanism.

Turney SG, Lichtman JW - PLoS Biol. (2012)

Time course of removal of laser-target input and takeover by another at developing neuromuscular junction.(A) In vivo image of region in a P7 mouse sternomastoid muscle. Boxed area shows a neuromuscular junction innervated by two axons (green, YFP-filled axons; red, α-bungarotoxin-tagged acetylcholine receptors). The axon on the right in the boxed region was irradiated with a mode-locked infrared laser at the site of the circle. (B) The same region of muscle reimaged 27 h later showing that the irradiated axon has completely disappeared. (C) Time-lapse imaging reveals that the remaining axon grows to occupy the sites that were vacated by the damaged axon (arrow points to site of irradiation). Within 0.5 h of irradiation the right axon's terminal branches swell and clearly reveal that the undamaged axon, that remained brightly fluorescent, terminated in a bulb. The swelling of the damaged axon deformed the shape of the intact axon's terminal bulb (panels at 0.8, 1, and 1.5 h). At 1.8 h, the bulb recovered its original shape, presumably because the damaged input had lost its turgor presumably due to membrane leakage. Over the next hour, the fluorescence in the damaged terminals became fainter, its axon fragmented (see 1.8 h), and it largely became invisible. Reimaging the junction 27 h after damage showed that the remaining axon branched to occupy many of the sites previously occupied by the laser irradiated axon. Scale bar, 20 µm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3383738&req=5

pbio-1001352-g001: Time course of removal of laser-target input and takeover by another at developing neuromuscular junction.(A) In vivo image of region in a P7 mouse sternomastoid muscle. Boxed area shows a neuromuscular junction innervated by two axons (green, YFP-filled axons; red, α-bungarotoxin-tagged acetylcholine receptors). The axon on the right in the boxed region was irradiated with a mode-locked infrared laser at the site of the circle. (B) The same region of muscle reimaged 27 h later showing that the irradiated axon has completely disappeared. (C) Time-lapse imaging reveals that the remaining axon grows to occupy the sites that were vacated by the damaged axon (arrow points to site of irradiation). Within 0.5 h of irradiation the right axon's terminal branches swell and clearly reveal that the undamaged axon, that remained brightly fluorescent, terminated in a bulb. The swelling of the damaged axon deformed the shape of the intact axon's terminal bulb (panels at 0.8, 1, and 1.5 h). At 1.8 h, the bulb recovered its original shape, presumably because the damaged input had lost its turgor presumably due to membrane leakage. Over the next hour, the fluorescence in the damaged terminals became fainter, its axon fragmented (see 1.8 h), and it largely became invisible. Reimaging the junction 27 h after damage showed that the remaining axon branched to occupy many of the sites previously occupied by the laser irradiated axon. Scale bar, 20 µm.
Mentions: Damage to axons typically evolved over 30–45 min and the whole process of axon removal required many hours. Even though we observed bleaching of the axon segment at the time of irradiation, evidence for structural damage only became apparent within 10–20 min (see Figures 1–3). Signs of axon damage included dramatic swelling of the axon distal to the site of laser focus and a progressive widening of the region of non-fluorescence both distal and proximal to the laser irradiation site. Presumably this loss of fluorescence is secondary to leakage of proteins from the cytoplasm at the damage site. This phase which typically lasted up to several hours was followed by the complete disappearance of the distal axon save for occasionally a few small disconnected fluorescent fragments that ultimately all disappeared by 10 h. In the proximal direction the damage initiated a die-back that was reminiscent both in time course and scale of “acute axonal degeneration” of damaged central axons [26]. Typically, the die-back stopped at the proximal branch point (Figure 2), although sometimes it extended anterogradely from the branch point to cause the disappearance of other terminal branches. If the fluorescence at the laser spot recovered after several minutes, that was an indication that the fluorescence in the axonal branch had been bleached but the axon was not seriously damaged because no subsequent changes were noted over the next half hour to hour, or the following day (see Materials and Methods for details).

Bottom Line: Indeed, during normal development we observed withdrawal followed by takeover.The stimulus for axon growth is not postsynaptic cell inactivity because axons grow into unoccupied sites even when target cells are functionally innervated.These results demonstrate competition at the synaptic level and enable us to provide a conceptual framework for understanding this form of synaptic plasticity.

View Article: PubMed Central - PubMed

Affiliation: Center for Brain Science and Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, United States of America. sturney@mcb.harvard.edu

ABSTRACT
During mammalian development, neuromuscular junctions and some other postsynaptic cells transition from multiple- to single-innervation as synaptic sites are exchanged between different axons. It is unclear whether one axon invades synaptic sites to drive off other inputs or alternatively axons expand their territory in response to sites vacated by other axons. Here we show that soon-to-be-eliminated axons rapidly reverse fate and grow to occupy vacant sites at a neuromuscular junction after laser removal of a stronger input. This reversal supports the idea that axons take over sites that were previously vacated. Indeed, during normal development we observed withdrawal followed by takeover. The stimulus for axon growth is not postsynaptic cell inactivity because axons grow into unoccupied sites even when target cells are functionally innervated. These results demonstrate competition at the synaptic level and enable us to provide a conceptual framework for understanding this form of synaptic plasticity.

Show MeSH
Related in: MedlinePlus