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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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Previously retracted axons reinnervate their former neuromuscular junctions after laser-targeted removal of the innervating axon.(A) Shown is a singly innervated neuromuscular junction with a nearby recently retracted axon (tipped by a bulb) in the sternomastoid muscle of P7 mouse in vivo. The pulsed laser irradiated the axon innervating the neuromuscular junction at the circle marked by a large arrow. After 1 h, the irradiated axon was mostly invisible, leaving the bulb-tipped retracting input unchanged. By the next day (1 d), the retracting input had reversed direction and nearly completely reinnervated the junction. After 2 d (2 d) the caliber of the reinnervating axon and its terminal branches increased. In this experiment it was unambiguous that the regenerated axon was the former withdrawing axon because the two axons could be distinguished by their relative concentrations of YFP and CFP. The bulb-tipped axon could also be re-identified after it reinnervated the junction because of the location of its proximal branch point (indicated by the small arrow in each image). Scale bar, 20 µm. (B) Graph showing the incidence of return of retracting axons as a function of its distance from the junction at the time of laser axotomy of the innervating axon (also see Table 1B). The percentages were computed relative to the total number of retracting axons studied. Retracting axons 10 µm or closer appeared more likely to return than ones farther away.
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pbio-1001352-g004: Previously retracted axons reinnervate their former neuromuscular junctions after laser-targeted removal of the innervating axon.(A) Shown is a singly innervated neuromuscular junction with a nearby recently retracted axon (tipped by a bulb) in the sternomastoid muscle of P7 mouse in vivo. The pulsed laser irradiated the axon innervating the neuromuscular junction at the circle marked by a large arrow. After 1 h, the irradiated axon was mostly invisible, leaving the bulb-tipped retracting input unchanged. By the next day (1 d), the retracting input had reversed direction and nearly completely reinnervated the junction. After 2 d (2 d) the caliber of the reinnervating axon and its terminal branches increased. In this experiment it was unambiguous that the regenerated axon was the former withdrawing axon because the two axons could be distinguished by their relative concentrations of YFP and CFP. The bulb-tipped axon could also be re-identified after it reinnervated the junction because of the location of its proximal branch point (indicated by the small arrow in each image). Scale bar, 20 µm. (B) Graph showing the incidence of return of retracting axons as a function of its distance from the junction at the time of laser axotomy of the innervating axon (also see Table 1B). The percentages were computed relative to the total number of retracting axons studied. Retracting axons 10 µm or closer appeared more likely to return than ones farther away.

Mentions: Once an axon has lost all territory at a neuromuscular junction it undergoes a stereotyped process of withdrawal in which the bulb tipped axon branch sheds some of its cytoplasm and appears to retract away from the junction [11],[18],[27]. These “retraction bulbs” are seen frequently in developing muscles at the time of synapse elimination but are not seen at all in adult muscles. Might these structures be irreversibly committed to retraction? In anesthetized mice we successfully damaged 18 strong axonal inputs of singly innervated junctions where a second axon had recently retracted but was still visible nearby. Previous time lapse studies indicate that retracted axons which were within ∼200 µm of a junction had disconnected at some point over the previous 48 h [4],[11]. After damaging the axonal input that innervated the junction, we allowed the animals to recover and waited to see if nearby retracted inputs ever attempted to return to the junction over the following days. To our surprise, in 55% of the cases (n = 10/18), the axon stopped retracting, grew back to the junction, and occupied the entire junctional area (Table 1B). The laser-irradiated axon typically died back to the proximal branch point. Although in most cases given the position of the two axons it was unambiguous that the damaged axon did not reinnervate the junction, a potential ambiguity could occur if the damaged axon rapidly reinnervated the junction while at the same time the retracting axon completely disappeared. In order to directly identify the re-growing axon, we used a doubly transgenic mouse in which individual neurons expressed different concentrations of cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) in each axon (see Materials and Methods). In this case, we found that the damaged axon (identified by its color) did not return over the next 48 h, whereas the retracted axon (unambiguously identified by its color and its site of exit from the nerve fascicle) reinnervated the junction within 24 h and increased in caliber over the next day (Figure 4A).


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)

Previously retracted axons reinnervate their former neuromuscular junctions after laser-targeted removal of the innervating axon.(A) Shown is a singly innervated neuromuscular junction with a nearby recently retracted axon (tipped by a bulb) in the sternomastoid muscle of P7 mouse in vivo. The pulsed laser irradiated the axon innervating the neuromuscular junction at the circle marked by a large arrow. After 1 h, the irradiated axon was mostly invisible, leaving the bulb-tipped retracting input unchanged. By the next day (1 d), the retracting input had reversed direction and nearly completely reinnervated the junction. After 2 d (2 d) the caliber of the reinnervating axon and its terminal branches increased. In this experiment it was unambiguous that the regenerated axon was the former withdrawing axon because the two axons could be distinguished by their relative concentrations of YFP and CFP. The bulb-tipped axon could also be re-identified after it reinnervated the junction because of the location of its proximal branch point (indicated by the small arrow in each image). Scale bar, 20 µm. (B) Graph showing the incidence of return of retracting axons as a function of its distance from the junction at the time of laser axotomy of the innervating axon (also see Table 1B). The percentages were computed relative to the total number of retracting axons studied. Retracting axons 10 µm or closer appeared more likely to return than ones farther away.
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Related In: Results  -  Collection

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pbio-1001352-g004: Previously retracted axons reinnervate their former neuromuscular junctions after laser-targeted removal of the innervating axon.(A) Shown is a singly innervated neuromuscular junction with a nearby recently retracted axon (tipped by a bulb) in the sternomastoid muscle of P7 mouse in vivo. The pulsed laser irradiated the axon innervating the neuromuscular junction at the circle marked by a large arrow. After 1 h, the irradiated axon was mostly invisible, leaving the bulb-tipped retracting input unchanged. By the next day (1 d), the retracting input had reversed direction and nearly completely reinnervated the junction. After 2 d (2 d) the caliber of the reinnervating axon and its terminal branches increased. In this experiment it was unambiguous that the regenerated axon was the former withdrawing axon because the two axons could be distinguished by their relative concentrations of YFP and CFP. The bulb-tipped axon could also be re-identified after it reinnervated the junction because of the location of its proximal branch point (indicated by the small arrow in each image). Scale bar, 20 µm. (B) Graph showing the incidence of return of retracting axons as a function of its distance from the junction at the time of laser axotomy of the innervating axon (also see Table 1B). The percentages were computed relative to the total number of retracting axons studied. Retracting axons 10 µm or closer appeared more likely to return than ones farther away.
Mentions: Once an axon has lost all territory at a neuromuscular junction it undergoes a stereotyped process of withdrawal in which the bulb tipped axon branch sheds some of its cytoplasm and appears to retract away from the junction [11],[18],[27]. These “retraction bulbs” are seen frequently in developing muscles at the time of synapse elimination but are not seen at all in adult muscles. Might these structures be irreversibly committed to retraction? In anesthetized mice we successfully damaged 18 strong axonal inputs of singly innervated junctions where a second axon had recently retracted but was still visible nearby. Previous time lapse studies indicate that retracted axons which were within ∼200 µm of a junction had disconnected at some point over the previous 48 h [4],[11]. After damaging the axonal input that innervated the junction, we allowed the animals to recover and waited to see if nearby retracted inputs ever attempted to return to the junction over the following days. To our surprise, in 55% of the cases (n = 10/18), the axon stopped retracting, grew back to the junction, and occupied the entire junctional area (Table 1B). The laser-irradiated axon typically died back to the proximal branch point. Although in most cases given the position of the two axons it was unambiguous that the damaged axon did not reinnervate the junction, a potential ambiguity could occur if the damaged axon rapidly reinnervated the junction while at the same time the retracting axon completely disappeared. In order to directly identify the re-growing axon, we used a doubly transgenic mouse in which individual neurons expressed different concentrations of cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) in each axon (see Materials and Methods). In this case, we found that the damaged axon (identified by its color) did not return over the next 48 h, whereas the retracted axon (unambiguously identified by its color and its site of exit from the nerve fascicle) reinnervated the junction within 24 h and increased in caliber over the next day (Figure 4A).

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