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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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NMJs shed ghost boutons that stabilize or disappear.(A) Example of live imaging of an NMJ through the cuticle of an intact larvae expressing channelrhodopsin-2 and mRFP (red) in motoneurons, and a synaptically targeted mCD8-Shaker-GFP protein (green) in postsynaptic muscles. Motor neurons were stimulated with a spaced blue light paradigm (as in Figure 1) and NMJs were imaged at indicated times. Stimulation led to the formation of a ghost bouton (arrow) that lacked postsynaptic mCD8-Shaker-GFP. 18 h later, the ghost bouton was eliminated. (B) Live, intact larvae expressing channelrhodopsin-2 and mCD8-GFP in motor neurons were imaged immediately and 4 h after spaced light stimulation. White arrows point to ghost boutons observed before and after stimulation. Black arrowheads point to presynaptic debris that formed after stimulation. (C–E) Live, intact larvae expressing channelrhodopsin-2 and mCD8-GFP in motor neurons were imaged immediately and at 1-h intervals after spaced light stimulation. In some instances, detached ghost boutons simply became smaller and disappeared leaving debris (C and D, arrows), while detached ghost boutons sometimes simply became smaller and disappeared without leaving any obvious debris (E, white arrows) Presynaptic debris at NMJ regions devoid of ghost boutons would also appear and then disappear following stimulation (E, black and pink arrowheads). Calibration scale is 17 µm for (A, and C–E), 12 µm for (B), and 9 µm for (A, inset). Times correspond to hours from beginning of experiment when preparations were first imaged.
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pbio-1000184-g002: NMJs shed ghost boutons that stabilize or disappear.(A) Example of live imaging of an NMJ through the cuticle of an intact larvae expressing channelrhodopsin-2 and mRFP (red) in motoneurons, and a synaptically targeted mCD8-Shaker-GFP protein (green) in postsynaptic muscles. Motor neurons were stimulated with a spaced blue light paradigm (as in Figure 1) and NMJs were imaged at indicated times. Stimulation led to the formation of a ghost bouton (arrow) that lacked postsynaptic mCD8-Shaker-GFP. 18 h later, the ghost bouton was eliminated. (B) Live, intact larvae expressing channelrhodopsin-2 and mCD8-GFP in motor neurons were imaged immediately and 4 h after spaced light stimulation. White arrows point to ghost boutons observed before and after stimulation. Black arrowheads point to presynaptic debris that formed after stimulation. (C–E) Live, intact larvae expressing channelrhodopsin-2 and mCD8-GFP in motor neurons were imaged immediately and at 1-h intervals after spaced light stimulation. In some instances, detached ghost boutons simply became smaller and disappeared leaving debris (C and D, arrows), while detached ghost boutons sometimes simply became smaller and disappeared without leaving any obvious debris (E, white arrows) Presynaptic debris at NMJ regions devoid of ghost boutons would also appear and then disappear following stimulation (E, black and pink arrowheads). Calibration scale is 17 µm for (A, and C–E), 12 µm for (B), and 9 µm for (A, inset). Times correspond to hours from beginning of experiment when preparations were first imaged.

Mentions: We also conducted time-lapse imaging of identified NMJs from live intact larvae expressing ChR2 in motorneurons using C380-Gal4 [20]. These larvae also contained fluorescent markers that allowed us to simultaneously image the pre- and the postsynaptic compartment. In particular, these larvae expressed UAS-mRFP in motorneurons to visualize the presynaptic NMJ arbor and mCD8-GFP::Sh in muscles using the myosin heavy chain (MHC) promoter [21] to visualize the postsynaptic NMJ region. In the MHC-mCD8-GFP::Sh transgene, the GFP is fused to the last ∼150 C-terminal amino acids of the Shaker K+ channel isoform containing a Discs-Large (DLG) PDZ binding site, and thus it is targeted to the postsynaptic region allowing its visualization in vivo [21]. These larvae were subjected to spaced stimulation with light as above, and the same NMJ imaged for 5–15 min at different intervals. Between imaging intervals larvae were returned to the food. As previously reported [13], we found that ghost boutons were present and some of these became stabilized and recruited postsynaptic label. However, we also observed that many of these ghost boutons did not recruit postsynaptic label and disappeared over time (Figure 2A, arrow and inset in right panel).


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)

NMJs shed ghost boutons that stabilize or disappear.(A) Example of live imaging of an NMJ through the cuticle of an intact larvae expressing channelrhodopsin-2 and mRFP (red) in motoneurons, and a synaptically targeted mCD8-Shaker-GFP protein (green) in postsynaptic muscles. Motor neurons were stimulated with a spaced blue light paradigm (as in Figure 1) and NMJs were imaged at indicated times. Stimulation led to the formation of a ghost bouton (arrow) that lacked postsynaptic mCD8-Shaker-GFP. 18 h later, the ghost bouton was eliminated. (B) Live, intact larvae expressing channelrhodopsin-2 and mCD8-GFP in motor neurons were imaged immediately and 4 h after spaced light stimulation. White arrows point to ghost boutons observed before and after stimulation. Black arrowheads point to presynaptic debris that formed after stimulation. (C–E) Live, intact larvae expressing channelrhodopsin-2 and mCD8-GFP in motor neurons were imaged immediately and at 1-h intervals after spaced light stimulation. In some instances, detached ghost boutons simply became smaller and disappeared leaving debris (C and D, arrows), while detached ghost boutons sometimes simply became smaller and disappeared without leaving any obvious debris (E, white arrows) Presynaptic debris at NMJ regions devoid of ghost boutons would also appear and then disappear following stimulation (E, black and pink arrowheads). Calibration scale is 17 µm for (A, and C–E), 12 µm for (B), and 9 µm for (A, inset). Times correspond to hours from beginning of experiment when preparations were first imaged.
© Copyright Policy
Related In: Results  -  Collection

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

pbio-1000184-g002: NMJs shed ghost boutons that stabilize or disappear.(A) Example of live imaging of an NMJ through the cuticle of an intact larvae expressing channelrhodopsin-2 and mRFP (red) in motoneurons, and a synaptically targeted mCD8-Shaker-GFP protein (green) in postsynaptic muscles. Motor neurons were stimulated with a spaced blue light paradigm (as in Figure 1) and NMJs were imaged at indicated times. Stimulation led to the formation of a ghost bouton (arrow) that lacked postsynaptic mCD8-Shaker-GFP. 18 h later, the ghost bouton was eliminated. (B) Live, intact larvae expressing channelrhodopsin-2 and mCD8-GFP in motor neurons were imaged immediately and 4 h after spaced light stimulation. White arrows point to ghost boutons observed before and after stimulation. Black arrowheads point to presynaptic debris that formed after stimulation. (C–E) Live, intact larvae expressing channelrhodopsin-2 and mCD8-GFP in motor neurons were imaged immediately and at 1-h intervals after spaced light stimulation. In some instances, detached ghost boutons simply became smaller and disappeared leaving debris (C and D, arrows), while detached ghost boutons sometimes simply became smaller and disappeared without leaving any obvious debris (E, white arrows) Presynaptic debris at NMJ regions devoid of ghost boutons would also appear and then disappear following stimulation (E, black and pink arrowheads). Calibration scale is 17 µm for (A, and C–E), 12 µm for (B), and 9 µm for (A, inset). Times correspond to hours from beginning of experiment when preparations were first imaged.
Mentions: We also conducted time-lapse imaging of identified NMJs from live intact larvae expressing ChR2 in motorneurons using C380-Gal4 [20]. These larvae also contained fluorescent markers that allowed us to simultaneously image the pre- and the postsynaptic compartment. In particular, these larvae expressed UAS-mRFP in motorneurons to visualize the presynaptic NMJ arbor and mCD8-GFP::Sh in muscles using the myosin heavy chain (MHC) promoter [21] to visualize the postsynaptic NMJ region. In the MHC-mCD8-GFP::Sh transgene, the GFP is fused to the last ∼150 C-terminal amino acids of the Shaker K+ channel isoform containing a Discs-Large (DLG) PDZ binding site, and thus it is targeted to the postsynaptic region allowing its visualization in vivo [21]. These larvae were subjected to spaced stimulation with light as above, and the same NMJ imaged for 5–15 min at different intervals. Between imaging intervals larvae were returned to the food. As previously reported [13], we found that ghost boutons were present and some of these became stabilized and recruited postsynaptic label. However, we also observed that many of these ghost boutons did not recruit postsynaptic label and disappeared over time (Figure 2A, arrow and inset in right panel).

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