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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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dCed-6, a key component of the Draper signaling pathway, is required for clearance of ghost boutons and presynaptic debris and for normal synaptic growth.dCed-6 function at the NMJ was assayed by expressing UAS-dCed-6-RNAi in glia, motor neurons, and muscles. Preparations were labeled with the presynaptic marker α-HRP (red) and the postsynaptic marker α-DLG (green). (Ai–ii) Wild-type NMJs exhibit little or no presynaptic debris and ghost boutons. (Bi–ii) Muscle-specific dCed-6 knockdown leads to the accumulation of ghost boutons (arrowheads) but very little presynaptic debris. (Ci–ii) Glial-specific dCed-6 knockdown leads to the accumulation of presynaptic debris (arrows) but not ghost boutons. (D–F) Quantification of the number of (D) type Ib boutons, (E) ghost boutons, and (F) presynaptic debris in control and dCed-6 knockdown backgrounds. dCed-6 function is required in both muscles and glia for (D) normal synaptic growth, in (E) muscles for the clearance of ghost boutons, and (F) in glia for clearance of presynaptic debris. ***, p<0.001; **, p≤0.01; *, p≤0.05. For (D–F), n = 12 for wild type, 9 for drprΔ5, and 13 for dCed-6RNAi. Calibration scale is 12 µm.
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pbio-1000184-g007: dCed-6, a key component of the Draper signaling pathway, is required for clearance of ghost boutons and presynaptic debris and for normal synaptic growth.dCed-6 function at the NMJ was assayed by expressing UAS-dCed-6-RNAi in glia, motor neurons, and muscles. Preparations were labeled with the presynaptic marker α-HRP (red) and the postsynaptic marker α-DLG (green). (Ai–ii) Wild-type NMJs exhibit little or no presynaptic debris and ghost boutons. (Bi–ii) Muscle-specific dCed-6 knockdown leads to the accumulation of ghost boutons (arrowheads) but very little presynaptic debris. (Ci–ii) Glial-specific dCed-6 knockdown leads to the accumulation of presynaptic debris (arrows) but not ghost boutons. (D–F) Quantification of the number of (D) type Ib boutons, (E) ghost boutons, and (F) presynaptic debris in control and dCed-6 knockdown backgrounds. dCed-6 function is required in both muscles and glia for (D) normal synaptic growth, in (E) muscles for the clearance of ghost boutons, and (F) in glia for clearance of presynaptic debris. ***, p<0.001; **, p≤0.01; *, p≤0.05. For (D–F), n = 12 for wild type, 9 for drprΔ5, and 13 for dCed-6RNAi. Calibration scale is 12 µm.

Mentions: Previous studies have shown that the PTB-domain protein dCed-6 functions downstream of Draper [23]. Therefore, we used RNAi knockdown of dCed-6 in muscle or glia as a second approach to blocking glial and muscle engulfment activity. As in draper mutants, downregulating dCed-6 in either muscle or peripheral glia resulted in significant decrease in the number of synaptic boutons (Figure 7A–7D). In contrast, no effect was observed when dCed-6-RNAi was expressed in motorneurons (Figure 7D). Similar to Draper RNAi knockdown, expressing dCed-6-RNAi in muscles or glia had differential consequences for the appearance of presynaptic debris versus ghost boutons. Decreased levels of dCed-6 in muscles led to an increase in the number of ghost boutons, but had no influence in the deposition of presynaptic debris (Figure 7B, 7E, and 7F). Downregulating dCed-6 in glia, on the other hand, led to a significant increase in presynaptic debris deposition, but the number of ghost boutons remained unaltered (Figure 7C, 7E, and 7F). These results are consistent with the notion that dCed-6 functions downstream of Draper during the development of the NMJ. Further, they support the model that both muscle and glia contribute differentially to the clearance of debris versus ghost boutons at the NMJ.


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)

dCed-6, a key component of the Draper signaling pathway, is required for clearance of ghost boutons and presynaptic debris and for normal synaptic growth.dCed-6 function at the NMJ was assayed by expressing UAS-dCed-6-RNAi in glia, motor neurons, and muscles. Preparations were labeled with the presynaptic marker α-HRP (red) and the postsynaptic marker α-DLG (green). (Ai–ii) Wild-type NMJs exhibit little or no presynaptic debris and ghost boutons. (Bi–ii) Muscle-specific dCed-6 knockdown leads to the accumulation of ghost boutons (arrowheads) but very little presynaptic debris. (Ci–ii) Glial-specific dCed-6 knockdown leads to the accumulation of presynaptic debris (arrows) but not ghost boutons. (D–F) Quantification of the number of (D) type Ib boutons, (E) ghost boutons, and (F) presynaptic debris in control and dCed-6 knockdown backgrounds. dCed-6 function is required in both muscles and glia for (D) normal synaptic growth, in (E) muscles for the clearance of ghost boutons, and (F) in glia for clearance of presynaptic debris. ***, p<0.001; **, p≤0.01; *, p≤0.05. For (D–F), n = 12 for wild type, 9 for drprΔ5, and 13 for dCed-6RNAi. Calibration scale is 12 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2724735&req=5

pbio-1000184-g007: dCed-6, a key component of the Draper signaling pathway, is required for clearance of ghost boutons and presynaptic debris and for normal synaptic growth.dCed-6 function at the NMJ was assayed by expressing UAS-dCed-6-RNAi in glia, motor neurons, and muscles. Preparations were labeled with the presynaptic marker α-HRP (red) and the postsynaptic marker α-DLG (green). (Ai–ii) Wild-type NMJs exhibit little or no presynaptic debris and ghost boutons. (Bi–ii) Muscle-specific dCed-6 knockdown leads to the accumulation of ghost boutons (arrowheads) but very little presynaptic debris. (Ci–ii) Glial-specific dCed-6 knockdown leads to the accumulation of presynaptic debris (arrows) but not ghost boutons. (D–F) Quantification of the number of (D) type Ib boutons, (E) ghost boutons, and (F) presynaptic debris in control and dCed-6 knockdown backgrounds. dCed-6 function is required in both muscles and glia for (D) normal synaptic growth, in (E) muscles for the clearance of ghost boutons, and (F) in glia for clearance of presynaptic debris. ***, p<0.001; **, p≤0.01; *, p≤0.05. For (D–F), n = 12 for wild type, 9 for drprΔ5, and 13 for dCed-6RNAi. Calibration scale is 12 µm.
Mentions: Previous studies have shown that the PTB-domain protein dCed-6 functions downstream of Draper [23]. Therefore, we used RNAi knockdown of dCed-6 in muscle or glia as a second approach to blocking glial and muscle engulfment activity. As in draper mutants, downregulating dCed-6 in either muscle or peripheral glia resulted in significant decrease in the number of synaptic boutons (Figure 7A–7D). In contrast, no effect was observed when dCed-6-RNAi was expressed in motorneurons (Figure 7D). Similar to Draper RNAi knockdown, expressing dCed-6-RNAi in muscles or glia had differential consequences for the appearance of presynaptic debris versus ghost boutons. Decreased levels of dCed-6 in muscles led to an increase in the number of ghost boutons, but had no influence in the deposition of presynaptic debris (Figure 7B, 7E, and 7F). Downregulating dCed-6 in glia, on the other hand, led to a significant increase in presynaptic debris deposition, but the number of ghost boutons remained unaltered (Figure 7C, 7E, and 7F). These results are consistent with the notion that dCed-6 functions downstream of Draper during the development of the NMJ. Further, they support the model that both muscle and glia contribute differentially to the clearance of debris versus ghost boutons at the NMJ.

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