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Glial processes at the Drosophila larval neuromuscular junction match synaptic growth.

Brink DL, Gilbert M, Xie X, Petley-Ragan L, Auld VJ - PLoS ONE (2012)

Bottom Line: Growth of the glial processes was coordinated with NMJ growth and synaptic activity.We found that elevated temperature was required during embryogenesis in order to increase glial expansion at the nmj.Therefore, in our live imaging system, glial processes at the NMJ are likely indirectly regulated by synaptic changes to ensure the coordinated growth of all components of the tripartite larval NMJ.

View Article: PubMed Central - PubMed

Affiliation: Department of Zoology, Cell and Developmental Biology, Life Sciences Institute, University of British Columbia, Vancouver, British Columbia, Canada.

ABSTRACT
Glia are integral participants in synaptic physiology, remodeling and maturation from blowflies to humans, yet how glial structure is coordinated with synaptic growth is unknown. To investigate the dynamics of glial development at the Drosophila larval neuromuscular junction (NMJ), we developed a live imaging system to establish the relationship between glia, neuronal boutons, and the muscle subsynaptic reticulum. Using this system we observed processes from two classes of peripheral glia present at the NMJ. Processes from the subperineurial glia formed a blood-nerve barrier around the axon proximal to the first bouton. Processes from the perineurial glial extended beyond the end of the blood-nerve barrier into the NMJ where they contacted synapses and extended across non-synaptic muscle. Growth of the glial processes was coordinated with NMJ growth and synaptic activity. Increasing synaptic size through elevated temperature or the highwire mutation increased the extent of glial processes at the NMJ and conversely blocking synaptic activity and size decreased the presence and size of glial processes. We found that elevated temperature was required during embryogenesis in order to increase glial expansion at the nmj. Therefore, in our live imaging system, glial processes at the NMJ are likely indirectly regulated by synaptic changes to ensure the coordinated growth of all components of the tripartite larval NMJ.

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Related in: MedlinePlus

Neurexin IV defined the motor axon end and the septate junction barrier proximal to the synaptic boutons.A–D) Fixed NMJ preparations where the septate junctions generated by the subperineurial glia (SPG) were labeled using Neurexin IV-GFP (NrxIV, green), the boutons and axons with anti-HRP (α-HRP, red) and the post-synaptic SSR with anti-Dlg (Dlg, blue). A 2D projection of the entire stack is shown in each panel. A) A fixed NMJ from a W3 larva with the corresponding grayscale showing the Neurexin IV-GFP. Scale bar, 15 µm. B–C) The boxed regions in panel A were digitally scaled 400% and the corresponding grayscale panels show the Neurexin IV-GFP distribution. Neurexin IV is excluded from synapses; limited to small axon branches and terminates prior to the first bouton. The septate junction termini formed blunt or tapered ends (B; arrowhead) with the occasion bulb-like structure (C; arrowhead). D) A fixed NMJ from a F3 larva in which the panels have been digitally scaled 300%. The septate junction continued along the axon from the root (asterisk) but stopped just before the proximal bouton of each branch (arrowheads). Scale bar, 5 µm. E–F) A live NMJ from a F3 larvae with the septate junctions labeled with Neurexin IV-GFP, the glia labeled using repo>CD8-RFP (red) and the neurons/boutons lived labeled using anti-HRP antibodies (blue). E) Fluorescently tagged anti-HRP antibody (α-HRP, blue) labeled the NMJ including those areas contacted by glial processes (repo>RFP, red) but the axons remain unlabeled (arrows) in the regions bounded by the septate junctions (NrxIV, green). The grayscale image shows the extent of anti-HRP antibody immunolabeling and the unlabeled axons (arrows). F, G) The boxed regions in panel E were digitally scaled 200%. Glial processes (arrows) labeled with RFP (repo>RFP) (I, J; red: Ii, Ji; magenta) extend beyond the terminus of the septate junction (NrxIV, green) (arrowheads).
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pone-0037876-g005: Neurexin IV defined the motor axon end and the septate junction barrier proximal to the synaptic boutons.A–D) Fixed NMJ preparations where the septate junctions generated by the subperineurial glia (SPG) were labeled using Neurexin IV-GFP (NrxIV, green), the boutons and axons with anti-HRP (α-HRP, red) and the post-synaptic SSR with anti-Dlg (Dlg, blue). A 2D projection of the entire stack is shown in each panel. A) A fixed NMJ from a W3 larva with the corresponding grayscale showing the Neurexin IV-GFP. Scale bar, 15 µm. B–C) The boxed regions in panel A were digitally scaled 400% and the corresponding grayscale panels show the Neurexin IV-GFP distribution. Neurexin IV is excluded from synapses; limited to small axon branches and terminates prior to the first bouton. The septate junction termini formed blunt or tapered ends (B; arrowhead) with the occasion bulb-like structure (C; arrowhead). D) A fixed NMJ from a F3 larva in which the panels have been digitally scaled 300%. The septate junction continued along the axon from the root (asterisk) but stopped just before the proximal bouton of each branch (arrowheads). Scale bar, 5 µm. E–F) A live NMJ from a F3 larvae with the septate junctions labeled with Neurexin IV-GFP, the glia labeled using repo>CD8-RFP (red) and the neurons/boutons lived labeled using anti-HRP antibodies (blue). E) Fluorescently tagged anti-HRP antibody (α-HRP, blue) labeled the NMJ including those areas contacted by glial processes (repo>RFP, red) but the axons remain unlabeled (arrows) in the regions bounded by the septate junctions (NrxIV, green). The grayscale image shows the extent of anti-HRP antibody immunolabeling and the unlabeled axons (arrows). F, G) The boxed regions in panel E were digitally scaled 200%. Glial processes (arrows) labeled with RFP (repo>RFP) (I, J; red: Ii, Ji; magenta) extend beyond the terminus of the septate junction (NrxIV, green) (arrowheads).

Mentions: Using repo-GAL4, Gliotactin-GAL4 and 46F-GAL4, we observed glial processes that extended beyond the septate junction into the synaptic region. To investigate the relationship between the blood-nerve barrier and the glial processes that extend into the synaptic region, we used Neurexin IV endogenously tagged with GFP (NrxIV-GFP) to visualize the septate junctions [2], [3], [4]. In both living and fixed tissues, NrxIV-GFP terminated on small axon branches proximal to the first synaptic boutons (Figure 5; Video S4). NrxIV-GFP was not located near the SSR immunolabeled with antibodies to Discs-large (Dlg) (Figure 5A–D) or with the boutons immunolabeled with anti-HRP (Figure 5A–G). The terminal ends of the septate junctions formed tapered lines (Figure 5B; arrowhead), blunt endings (Figure 5D: arrowhead) or bulb-like structures (Figure 5C: arrowhead). The NrxIV-GFP distribution suggested that the septate junctions form a permeability barrier at the motor axon end just prior to the first synaptic bouton. To functionally test for the presence of a diffusion barrier, we labeled live larval preparations expressing NrxIV-GFP with a fluorescently tagged primary antibody against HRP (Figure 5E–G). The antibody labeling was absent from the regions bounded by NrxIV-GFP (Figure 5F, G: arrows) and commenced distal to the NrxIV-GFP terminus (Figure 5F, G: arrowheads), confirming the antibody was excluded from glial structures expressing NrxIV. In contrast, the glial processes that extend into the NMJ did not exclude the anti-HRP antibody from labeling nearby boutons (Figure 5F, G: arrows). These results indicate that barrier function terminates at the septate junction termini labeled with NrxIV and is not a general property of glial processes within the NMJ proper.


Glial processes at the Drosophila larval neuromuscular junction match synaptic growth.

Brink DL, Gilbert M, Xie X, Petley-Ragan L, Auld VJ - PLoS ONE (2012)

Neurexin IV defined the motor axon end and the septate junction barrier proximal to the synaptic boutons.A–D) Fixed NMJ preparations where the septate junctions generated by the subperineurial glia (SPG) were labeled using Neurexin IV-GFP (NrxIV, green), the boutons and axons with anti-HRP (α-HRP, red) and the post-synaptic SSR with anti-Dlg (Dlg, blue). A 2D projection of the entire stack is shown in each panel. A) A fixed NMJ from a W3 larva with the corresponding grayscale showing the Neurexin IV-GFP. Scale bar, 15 µm. B–C) The boxed regions in panel A were digitally scaled 400% and the corresponding grayscale panels show the Neurexin IV-GFP distribution. Neurexin IV is excluded from synapses; limited to small axon branches and terminates prior to the first bouton. The septate junction termini formed blunt or tapered ends (B; arrowhead) with the occasion bulb-like structure (C; arrowhead). D) A fixed NMJ from a F3 larva in which the panels have been digitally scaled 300%. The septate junction continued along the axon from the root (asterisk) but stopped just before the proximal bouton of each branch (arrowheads). Scale bar, 5 µm. E–F) A live NMJ from a F3 larvae with the septate junctions labeled with Neurexin IV-GFP, the glia labeled using repo>CD8-RFP (red) and the neurons/boutons lived labeled using anti-HRP antibodies (blue). E) Fluorescently tagged anti-HRP antibody (α-HRP, blue) labeled the NMJ including those areas contacted by glial processes (repo>RFP, red) but the axons remain unlabeled (arrows) in the regions bounded by the septate junctions (NrxIV, green). The grayscale image shows the extent of anti-HRP antibody immunolabeling and the unlabeled axons (arrows). F, G) The boxed regions in panel E were digitally scaled 200%. Glial processes (arrows) labeled with RFP (repo>RFP) (I, J; red: Ii, Ji; magenta) extend beyond the terminus of the septate junction (NrxIV, green) (arrowheads).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3362601&req=5

pone-0037876-g005: Neurexin IV defined the motor axon end and the septate junction barrier proximal to the synaptic boutons.A–D) Fixed NMJ preparations where the septate junctions generated by the subperineurial glia (SPG) were labeled using Neurexin IV-GFP (NrxIV, green), the boutons and axons with anti-HRP (α-HRP, red) and the post-synaptic SSR with anti-Dlg (Dlg, blue). A 2D projection of the entire stack is shown in each panel. A) A fixed NMJ from a W3 larva with the corresponding grayscale showing the Neurexin IV-GFP. Scale bar, 15 µm. B–C) The boxed regions in panel A were digitally scaled 400% and the corresponding grayscale panels show the Neurexin IV-GFP distribution. Neurexin IV is excluded from synapses; limited to small axon branches and terminates prior to the first bouton. The septate junction termini formed blunt or tapered ends (B; arrowhead) with the occasion bulb-like structure (C; arrowhead). D) A fixed NMJ from a F3 larva in which the panels have been digitally scaled 300%. The septate junction continued along the axon from the root (asterisk) but stopped just before the proximal bouton of each branch (arrowheads). Scale bar, 5 µm. E–F) A live NMJ from a F3 larvae with the septate junctions labeled with Neurexin IV-GFP, the glia labeled using repo>CD8-RFP (red) and the neurons/boutons lived labeled using anti-HRP antibodies (blue). E) Fluorescently tagged anti-HRP antibody (α-HRP, blue) labeled the NMJ including those areas contacted by glial processes (repo>RFP, red) but the axons remain unlabeled (arrows) in the regions bounded by the septate junctions (NrxIV, green). The grayscale image shows the extent of anti-HRP antibody immunolabeling and the unlabeled axons (arrows). F, G) The boxed regions in panel E were digitally scaled 200%. Glial processes (arrows) labeled with RFP (repo>RFP) (I, J; red: Ii, Ji; magenta) extend beyond the terminus of the septate junction (NrxIV, green) (arrowheads).
Mentions: Using repo-GAL4, Gliotactin-GAL4 and 46F-GAL4, we observed glial processes that extended beyond the septate junction into the synaptic region. To investigate the relationship between the blood-nerve barrier and the glial processes that extend into the synaptic region, we used Neurexin IV endogenously tagged with GFP (NrxIV-GFP) to visualize the septate junctions [2], [3], [4]. In both living and fixed tissues, NrxIV-GFP terminated on small axon branches proximal to the first synaptic boutons (Figure 5; Video S4). NrxIV-GFP was not located near the SSR immunolabeled with antibodies to Discs-large (Dlg) (Figure 5A–D) or with the boutons immunolabeled with anti-HRP (Figure 5A–G). The terminal ends of the septate junctions formed tapered lines (Figure 5B; arrowhead), blunt endings (Figure 5D: arrowhead) or bulb-like structures (Figure 5C: arrowhead). The NrxIV-GFP distribution suggested that the septate junctions form a permeability barrier at the motor axon end just prior to the first synaptic bouton. To functionally test for the presence of a diffusion barrier, we labeled live larval preparations expressing NrxIV-GFP with a fluorescently tagged primary antibody against HRP (Figure 5E–G). The antibody labeling was absent from the regions bounded by NrxIV-GFP (Figure 5F, G: arrows) and commenced distal to the NrxIV-GFP terminus (Figure 5F, G: arrowheads), confirming the antibody was excluded from glial structures expressing NrxIV. In contrast, the glial processes that extend into the NMJ did not exclude the anti-HRP antibody from labeling nearby boutons (Figure 5F, G: arrows). These results indicate that barrier function terminates at the septate junction termini labeled with NrxIV and is not a general property of glial processes within the NMJ proper.

Bottom Line: Growth of the glial processes was coordinated with NMJ growth and synaptic activity.We found that elevated temperature was required during embryogenesis in order to increase glial expansion at the nmj.Therefore, in our live imaging system, glial processes at the NMJ are likely indirectly regulated by synaptic changes to ensure the coordinated growth of all components of the tripartite larval NMJ.

View Article: PubMed Central - PubMed

Affiliation: Department of Zoology, Cell and Developmental Biology, Life Sciences Institute, University of British Columbia, Vancouver, British Columbia, Canada.

ABSTRACT
Glia are integral participants in synaptic physiology, remodeling and maturation from blowflies to humans, yet how glial structure is coordinated with synaptic growth is unknown. To investigate the dynamics of glial development at the Drosophila larval neuromuscular junction (NMJ), we developed a live imaging system to establish the relationship between glia, neuronal boutons, and the muscle subsynaptic reticulum. Using this system we observed processes from two classes of peripheral glia present at the NMJ. Processes from the subperineurial glia formed a blood-nerve barrier around the axon proximal to the first bouton. Processes from the perineurial glial extended beyond the end of the blood-nerve barrier into the NMJ where they contacted synapses and extended across non-synaptic muscle. Growth of the glial processes was coordinated with NMJ growth and synaptic activity. Increasing synaptic size through elevated temperature or the highwire mutation increased the extent of glial processes at the NMJ and conversely blocking synaptic activity and size decreased the presence and size of glial processes. We found that elevated temperature was required during embryogenesis in order to increase glial expansion at the nmj. Therefore, in our live imaging system, glial processes at the NMJ are likely indirectly regulated by synaptic changes to ensure the coordinated growth of all components of the tripartite larval NMJ.

Show MeSH
Related in: MedlinePlus