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Glia and muscle sculpt neuromuscular arbors by engulfing destabilized synaptic boutons and shed presynaptic debris.

Fuentes-Medel Y, Logan MA, Ashley J, Ataman B, Budnik V, Freeman MR - PLoS Biol. (2009)

Bottom Line: Interestingly, we find that glia dynamically invade the NMJ and, working together with muscle cells, phagocytose shed presynaptic material.Suppressing engulfment activity in glia or muscle by disrupting the Draper/Ced-6 pathway results in a dramatic accumulation of presynaptic debris, and synaptic growth in turn is severely compromised.Thus actively growing NMJ arbors appear to constitutively generate an excessive number of immature boutons, eliminate those that are not stabilized through a shedding process, and normal synaptic expansion requires the continuous clearance of this material by both glia and muscle cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, University of Massachusetts Medical School, Worcester, Massachusetts, USA.

ABSTRACT
Synapse remodeling is an extremely dynamic process, often regulated by neural activity. Here we show during activity-dependent synaptic growth at the Drosophila NMJ many immature synaptic boutons fail to form stable postsynaptic contacts, are selectively shed from the parent arbor, and degenerate or disappear from the neuromuscular junction (NMJ). Surprisingly, we also observe the widespread appearance of presynaptically derived "debris" during normal synaptic growth. The shedding of both immature boutons and presynaptic debris is enhanced by high-frequency stimulation of motorneurons, indicating that their formation is modulated by neural activity. Interestingly, we find that glia dynamically invade the NMJ and, working together with muscle cells, phagocytose shed presynaptic material. Suppressing engulfment activity in glia or muscle by disrupting the Draper/Ced-6 pathway results in a dramatic accumulation of presynaptic debris, and synaptic growth in turn is severely compromised. Thus actively growing NMJ arbors appear to constitutively generate an excessive number of immature boutons, eliminate those that are not stabilized through a shedding process, and normal synaptic expansion requires the continuous clearance of this material by both glia and muscle cells.

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Draper function is essential in both glia and muscle cells for clearance of ghost boutons and shed presynaptic debris and for normal synaptic growth.Draper function was knocked-down by expressing UAS-Draper-RNAi in either muscle (C57-Gal4), glia (repo-Gal4), or motor neurons (OK6-Gal4), and ghost boutons and presynaptic debris were quantified by staining for HRP (red), and the postsynapse was visualized with DLG (green). (Ai–ii) Wild-type NMJs have very little presynaptic debris and few or no ghost boutons. (Bi–ii) Muscle-specific Draper knockdown leads to the accumulation of ghost boutons (arrows), but not of presynaptic debris. (Ci–ii) Glial-specific Draper knockdown leads to the accumulation of presynaptic debris (arrows), but not of ghost boutons. (D) mCD8-GFP (green) was expressed in glia with repo-Gal4 and motor neurons were visualized by staining for HRP (red). representative images of weak HRP signal detected within glial extensions (arrowheads). (E) Quantification of number of type Ib synaptic boutons at muscle 6/7 showing that Draper knockdown in glia or muscle cells reduces bouton number to those in draperΔ5  mutants, while Draper knockdown in motor neurons has no effect. (F) Quantification of ghost bouton number. Knockdown of Draper in muscle cells, but not glia or motor neurons, leads to the accumulation of ghost boutons at levels equivalent to those found in draperΔ5  mutants. (G) Quantification of shed presynaptic debris. Draper knockdown in glial cells, but not muscles or motorneurons, leads to the accumulation of presynaptic debris at levels similar to draperΔ5  mutants. ***, p<0.001; **, p≤0.01; *, p≤0.05. Calibration scale is 12 µm for (A and B), and 3 µm for (D). (n≥10 for each genotype).
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pbio-1000184-g006: Draper function is essential in both glia and muscle cells for clearance of ghost boutons and shed presynaptic debris and for normal synaptic growth.Draper function was knocked-down by expressing UAS-Draper-RNAi in either muscle (C57-Gal4), glia (repo-Gal4), or motor neurons (OK6-Gal4), and ghost boutons and presynaptic debris were quantified by staining for HRP (red), and the postsynapse was visualized with DLG (green). (Ai–ii) Wild-type NMJs have very little presynaptic debris and few or no ghost boutons. (Bi–ii) Muscle-specific Draper knockdown leads to the accumulation of ghost boutons (arrows), but not of presynaptic debris. (Ci–ii) Glial-specific Draper knockdown leads to the accumulation of presynaptic debris (arrows), but not of ghost boutons. (D) mCD8-GFP (green) was expressed in glia with repo-Gal4 and motor neurons were visualized by staining for HRP (red). representative images of weak HRP signal detected within glial extensions (arrowheads). (E) Quantification of number of type Ib synaptic boutons at muscle 6/7 showing that Draper knockdown in glia or muscle cells reduces bouton number to those in draperΔ5 mutants, while Draper knockdown in motor neurons has no effect. (F) Quantification of ghost bouton number. Knockdown of Draper in muscle cells, but not glia or motor neurons, leads to the accumulation of ghost boutons at levels equivalent to those found in draperΔ5 mutants. (G) Quantification of shed presynaptic debris. Draper knockdown in glial cells, but not muscles or motorneurons, leads to the accumulation of presynaptic debris at levels similar to draperΔ5 mutants. ***, p<0.001; **, p≤0.01; *, p≤0.05. Calibration scale is 12 µm for (A and B), and 3 µm for (D). (n≥10 for each genotype).

Mentions: To address the possibility that Draper might function both in glia and muscle to sculpt the NMJ we selectively expressed a Draper-RNAi designed to knockdown all Draper isoforms in glia or muscles using cell-specific Gal4 strains. RNAi knockdown of Draper in either muscle or glia resulted in a reduction in the number of synaptic boutons, which was not significantly different from the draper mutant (Figure 6E). This indicates that the removal of Draper from either cell type is sufficient to interfere with NMJ growth. Remarkably, however, downregulating Draper in muscle versus glia had a different consequence for the deposition of presynaptic debris and the appearance of detached ghost boutons. RNAi knockdown of Draper in glia resulted in an increase in presynaptic debris to an extent similar to the draper mutant (Figure 6C and 6G). However, no significant increase in the number of detached ghost boutons was observed (Figure 6F). If glial extensions are primarily involved in engulfing presynaptic debris, we predicted that we should find HRP positive debris within the glial extensions. We found that this was indeed the case. We found several instances in which glial terminals formed bulb-like structures that contained anti-HRP immunoreactive puncta within (Figure 6D, arrowheads).


Glia and muscle sculpt neuromuscular arbors by engulfing destabilized synaptic boutons and shed presynaptic debris.

Fuentes-Medel Y, Logan MA, Ashley J, Ataman B, Budnik V, Freeman MR - PLoS Biol. (2009)

Draper function is essential in both glia and muscle cells for clearance of ghost boutons and shed presynaptic debris and for normal synaptic growth.Draper function was knocked-down by expressing UAS-Draper-RNAi in either muscle (C57-Gal4), glia (repo-Gal4), or motor neurons (OK6-Gal4), and ghost boutons and presynaptic debris were quantified by staining for HRP (red), and the postsynapse was visualized with DLG (green). (Ai–ii) Wild-type NMJs have very little presynaptic debris and few or no ghost boutons. (Bi–ii) Muscle-specific Draper knockdown leads to the accumulation of ghost boutons (arrows), but not of presynaptic debris. (Ci–ii) Glial-specific Draper knockdown leads to the accumulation of presynaptic debris (arrows), but not of ghost boutons. (D) mCD8-GFP (green) was expressed in glia with repo-Gal4 and motor neurons were visualized by staining for HRP (red). representative images of weak HRP signal detected within glial extensions (arrowheads). (E) Quantification of number of type Ib synaptic boutons at muscle 6/7 showing that Draper knockdown in glia or muscle cells reduces bouton number to those in draperΔ5  mutants, while Draper knockdown in motor neurons has no effect. (F) Quantification of ghost bouton number. Knockdown of Draper in muscle cells, but not glia or motor neurons, leads to the accumulation of ghost boutons at levels equivalent to those found in draperΔ5  mutants. (G) Quantification of shed presynaptic debris. Draper knockdown in glial cells, but not muscles or motorneurons, leads to the accumulation of presynaptic debris at levels similar to draperΔ5  mutants. ***, p<0.001; **, p≤0.01; *, p≤0.05. Calibration scale is 12 µm for (A and B), and 3 µm for (D). (n≥10 for each genotype).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2724735&req=5

pbio-1000184-g006: Draper function is essential in both glia and muscle cells for clearance of ghost boutons and shed presynaptic debris and for normal synaptic growth.Draper function was knocked-down by expressing UAS-Draper-RNAi in either muscle (C57-Gal4), glia (repo-Gal4), or motor neurons (OK6-Gal4), and ghost boutons and presynaptic debris were quantified by staining for HRP (red), and the postsynapse was visualized with DLG (green). (Ai–ii) Wild-type NMJs have very little presynaptic debris and few or no ghost boutons. (Bi–ii) Muscle-specific Draper knockdown leads to the accumulation of ghost boutons (arrows), but not of presynaptic debris. (Ci–ii) Glial-specific Draper knockdown leads to the accumulation of presynaptic debris (arrows), but not of ghost boutons. (D) mCD8-GFP (green) was expressed in glia with repo-Gal4 and motor neurons were visualized by staining for HRP (red). representative images of weak HRP signal detected within glial extensions (arrowheads). (E) Quantification of number of type Ib synaptic boutons at muscle 6/7 showing that Draper knockdown in glia or muscle cells reduces bouton number to those in draperΔ5 mutants, while Draper knockdown in motor neurons has no effect. (F) Quantification of ghost bouton number. Knockdown of Draper in muscle cells, but not glia or motor neurons, leads to the accumulation of ghost boutons at levels equivalent to those found in draperΔ5 mutants. (G) Quantification of shed presynaptic debris. Draper knockdown in glial cells, but not muscles or motorneurons, leads to the accumulation of presynaptic debris at levels similar to draperΔ5 mutants. ***, p<0.001; **, p≤0.01; *, p≤0.05. Calibration scale is 12 µm for (A and B), and 3 µm for (D). (n≥10 for each genotype).
Mentions: To address the possibility that Draper might function both in glia and muscle to sculpt the NMJ we selectively expressed a Draper-RNAi designed to knockdown all Draper isoforms in glia or muscles using cell-specific Gal4 strains. RNAi knockdown of Draper in either muscle or glia resulted in a reduction in the number of synaptic boutons, which was not significantly different from the draper mutant (Figure 6E). This indicates that the removal of Draper from either cell type is sufficient to interfere with NMJ growth. Remarkably, however, downregulating Draper in muscle versus glia had a different consequence for the deposition of presynaptic debris and the appearance of detached ghost boutons. RNAi knockdown of Draper in glia resulted in an increase in presynaptic debris to an extent similar to the draper mutant (Figure 6C and 6G). However, no significant increase in the number of detached ghost boutons was observed (Figure 6F). If glial extensions are primarily involved in engulfing presynaptic debris, we predicted that we should find HRP positive debris within the glial extensions. We found that this was indeed the case. We found several instances in which glial terminals formed bulb-like structures that contained anti-HRP immunoreactive puncta within (Figure 6D, arrowheads).

Bottom Line: Interestingly, we find that glia dynamically invade the NMJ and, working together with muscle cells, phagocytose shed presynaptic material.Suppressing engulfment activity in glia or muscle by disrupting the Draper/Ced-6 pathway results in a dramatic accumulation of presynaptic debris, and synaptic growth in turn is severely compromised.Thus actively growing NMJ arbors appear to constitutively generate an excessive number of immature boutons, eliminate those that are not stabilized through a shedding process, and normal synaptic expansion requires the continuous clearance of this material by both glia and muscle cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, University of Massachusetts Medical School, Worcester, Massachusetts, USA.

ABSTRACT
Synapse remodeling is an extremely dynamic process, often regulated by neural activity. Here we show during activity-dependent synaptic growth at the Drosophila NMJ many immature synaptic boutons fail to form stable postsynaptic contacts, are selectively shed from the parent arbor, and degenerate or disappear from the neuromuscular junction (NMJ). Surprisingly, we also observe the widespread appearance of presynaptically derived "debris" during normal synaptic growth. The shedding of both immature boutons and presynaptic debris is enhanced by high-frequency stimulation of motorneurons, indicating that their formation is modulated by neural activity. Interestingly, we find that glia dynamically invade the NMJ and, working together with muscle cells, phagocytose shed presynaptic material. Suppressing engulfment activity in glia or muscle by disrupting the Draper/Ced-6 pathway results in a dramatic accumulation of presynaptic debris, and synaptic growth in turn is severely compromised. Thus actively growing NMJ arbors appear to constitutively generate an excessive number of immature boutons, eliminate those that are not stabilized through a shedding process, and normal synaptic expansion requires the continuous clearance of this material by both glia and muscle cells.

Show MeSH