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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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Increased rearing temperature expanded the perineurial glial processes.A–D) Live NMJs from F3 larvae with glial processes labeled using 46F>CD8-GFP (green), axons plus boutons immunolabeled with anti-HRP (red) and the SSR labeled with ShCter-DsRed (blue). Scale bars, 15 µm. The boxed regions (i) were digitally scaled 400% and all three channels shown in grayscale. A–B) NMJs from larvae raised at 18°C. Ai–Bi) Glial processes were small and most stopped at the axon end prior to the proximal boutons in the region corresponding to the end of the blood-nerve barrier (arrowheads). Glial areas: A = 15.7 µm2; B = 14.0 µm2 C–D) NMJs from larvae grown at 30°C. Ci–Di) Glial processes were more extensive and processes occupied a central channel along the synapse (arrows), plus extended across the muscle away from the NMJ (concave arrowhead). Glial areas: C = 16.0 µm2; D = 20.1 µm2 E–G) Glial membrane areas were measured and compared to neuron/bouton area (anti-HRP) and SSR area at 18°C, 25°C and 30°C. E) The mean area of glial processes (green) was significantly higher in larvae reared at 30°C (diamonds) than 18°C (circles) or 25°C (squares) (P<0.0002). Black symbols represent NMJs with glial processes that stopped at the motor axon end prior to the first bouton in the region defined as the terminus of the blood-nerve barrier. F) The mean ratio of glial process (GP) to neuron/bouton area (anti-HRP)(blue) was significantly greater at 30°C (diamonds) compared to 18°C (circles) and 25°C (squares) (P<0.0125). G) The mean ratio of GP to SSR area (magenta) was significantly greater for larvae reared at 30°C (diamonds) compared to 18°C (circles) and 25°C (squares) (P<0.0302).
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pone-0037876-g006: Increased rearing temperature expanded the perineurial glial processes.A–D) Live NMJs from F3 larvae with glial processes labeled using 46F>CD8-GFP (green), axons plus boutons immunolabeled with anti-HRP (red) and the SSR labeled with ShCter-DsRed (blue). Scale bars, 15 µm. The boxed regions (i) were digitally scaled 400% and all three channels shown in grayscale. A–B) NMJs from larvae raised at 18°C. Ai–Bi) Glial processes were small and most stopped at the axon end prior to the proximal boutons in the region corresponding to the end of the blood-nerve barrier (arrowheads). Glial areas: A = 15.7 µm2; B = 14.0 µm2 C–D) NMJs from larvae grown at 30°C. Ci–Di) Glial processes were more extensive and processes occupied a central channel along the synapse (arrows), plus extended across the muscle away from the NMJ (concave arrowhead). Glial areas: C = 16.0 µm2; D = 20.1 µm2 E–G) Glial membrane areas were measured and compared to neuron/bouton area (anti-HRP) and SSR area at 18°C, 25°C and 30°C. E) The mean area of glial processes (green) was significantly higher in larvae reared at 30°C (diamonds) than 18°C (circles) or 25°C (squares) (P<0.0002). Black symbols represent NMJs with glial processes that stopped at the motor axon end prior to the first bouton in the region defined as the terminus of the blood-nerve barrier. F) The mean ratio of glial process (GP) to neuron/bouton area (anti-HRP)(blue) was significantly greater at 30°C (diamonds) compared to 18°C (circles) and 25°C (squares) (P<0.0125). G) The mean ratio of GP to SSR area (magenta) was significantly greater for larvae reared at 30°C (diamonds) compared to 18°C (circles) and 25°C (squares) (P<0.0302).

Mentions: To test if glial processes at the NMJ responded to elevated temperature, we compared glial processes from feeding third instar larvae reared at three temperatures: 18, 25, and 30°C. We found that the GFP labeled processes were larger and had a higher frequency of expansion into synaptic regions at elevated temperatures (Figure 6). In addition, the type of glial processes qualitatively changed with elevated temperature. Profiles most prevalent at 18°C were blunt, shortened and terminated at the end of the motor axon in the region defined by the septate junction terminus (Figure 6A, B: arrowheads). At elevated temperatures glial processes extended further into the synapse, with more frequent processes in the synaptic region (Figure 6C, D: arrows). Occasionally the glial processes extended across the muscle, independent from the labeled boutons and SSR (Figure 6D: concave arrowhead).


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)

Increased rearing temperature expanded the perineurial glial processes.A–D) Live NMJs from F3 larvae with glial processes labeled using 46F>CD8-GFP (green), axons plus boutons immunolabeled with anti-HRP (red) and the SSR labeled with ShCter-DsRed (blue). Scale bars, 15 µm. The boxed regions (i) were digitally scaled 400% and all three channels shown in grayscale. A–B) NMJs from larvae raised at 18°C. Ai–Bi) Glial processes were small and most stopped at the axon end prior to the proximal boutons in the region corresponding to the end of the blood-nerve barrier (arrowheads). Glial areas: A = 15.7 µm2; B = 14.0 µm2 C–D) NMJs from larvae grown at 30°C. Ci–Di) Glial processes were more extensive and processes occupied a central channel along the synapse (arrows), plus extended across the muscle away from the NMJ (concave arrowhead). Glial areas: C = 16.0 µm2; D = 20.1 µm2 E–G) Glial membrane areas were measured and compared to neuron/bouton area (anti-HRP) and SSR area at 18°C, 25°C and 30°C. E) The mean area of glial processes (green) was significantly higher in larvae reared at 30°C (diamonds) than 18°C (circles) or 25°C (squares) (P<0.0002). Black symbols represent NMJs with glial processes that stopped at the motor axon end prior to the first bouton in the region defined as the terminus of the blood-nerve barrier. F) The mean ratio of glial process (GP) to neuron/bouton area (anti-HRP)(blue) was significantly greater at 30°C (diamonds) compared to 18°C (circles) and 25°C (squares) (P<0.0125). G) The mean ratio of GP to SSR area (magenta) was significantly greater for larvae reared at 30°C (diamonds) compared to 18°C (circles) and 25°C (squares) (P<0.0302).
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Related In: Results  -  Collection

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pone-0037876-g006: Increased rearing temperature expanded the perineurial glial processes.A–D) Live NMJs from F3 larvae with glial processes labeled using 46F>CD8-GFP (green), axons plus boutons immunolabeled with anti-HRP (red) and the SSR labeled with ShCter-DsRed (blue). Scale bars, 15 µm. The boxed regions (i) were digitally scaled 400% and all three channels shown in grayscale. A–B) NMJs from larvae raised at 18°C. Ai–Bi) Glial processes were small and most stopped at the axon end prior to the proximal boutons in the region corresponding to the end of the blood-nerve barrier (arrowheads). Glial areas: A = 15.7 µm2; B = 14.0 µm2 C–D) NMJs from larvae grown at 30°C. Ci–Di) Glial processes were more extensive and processes occupied a central channel along the synapse (arrows), plus extended across the muscle away from the NMJ (concave arrowhead). Glial areas: C = 16.0 µm2; D = 20.1 µm2 E–G) Glial membrane areas were measured and compared to neuron/bouton area (anti-HRP) and SSR area at 18°C, 25°C and 30°C. E) The mean area of glial processes (green) was significantly higher in larvae reared at 30°C (diamonds) than 18°C (circles) or 25°C (squares) (P<0.0002). Black symbols represent NMJs with glial processes that stopped at the motor axon end prior to the first bouton in the region defined as the terminus of the blood-nerve barrier. F) The mean ratio of glial process (GP) to neuron/bouton area (anti-HRP)(blue) was significantly greater at 30°C (diamonds) compared to 18°C (circles) and 25°C (squares) (P<0.0125). G) The mean ratio of GP to SSR area (magenta) was significantly greater for larvae reared at 30°C (diamonds) compared to 18°C (circles) and 25°C (squares) (P<0.0302).
Mentions: To test if glial processes at the NMJ responded to elevated temperature, we compared glial processes from feeding third instar larvae reared at three temperatures: 18, 25, and 30°C. We found that the GFP labeled processes were larger and had a higher frequency of expansion into synaptic regions at elevated temperatures (Figure 6). In addition, the type of glial processes qualitatively changed with elevated temperature. Profiles most prevalent at 18°C were blunt, shortened and terminated at the end of the motor axon in the region defined by the septate junction terminus (Figure 6A, B: arrowheads). At elevated temperatures glial processes extended further into the synapse, with more frequent processes in the synaptic region (Figure 6C, D: arrows). Occasionally the glial processes extended across the muscle, independent from the labeled boutons and SSR (Figure 6D: concave arrowhead).

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