Figure 5.
Figure 5. Axotomy depolarizes mitochondria in the vicinity of injured sites. (A) Representative images showing axonal mitochondria in adult DRG neurons before and after laser-based axotomy. Adult DRG neurons isolated from P60 mice were cotransfected with DsRed-Mito and GFP. Note that mitochondria at the axotomized site were immediately lost during axotomy (white arrows). (B and C) Kymographs showing axonal mitochondria labeled by DsRed-Mito (B) or colabeled by GFP-Mito and TMRE (C) before and after axotomy. The white arrows indicate laser-scanning sites (injured site), and the black arrows in the y axis show the laser execution time. Time-lapse images were captured to show that mitochondria in the vicinity suddenly shrunk (B) and lost the Δψm (TMRE staining; C). The images were first recorded at 5-s intervals for a total of 50 frames; the consecutive post-axotomy recording was collected at 5-s intervals for a total of 50 frames. (D and E) Representative images showing axotomy-induced depolarization of axonal mitochondria. Neurons were infected with pLenti-GFP (D) or GFP-Mito (E), and axons in the terminal chambers were loaded with 25 nM TMRE dye at DIV12, followed by laser-based axotomy and time-lapse imaging. Note that in neurons expressing GFP (D), axons were quickly broken up upon axotomy (white dashed lines), and a majority of mitochondria in the vicinity suddenly lost their TMRE staining (bottom right). In neurons expressing GFP-Mito (E), axotomy triggered a sudden loss of mitochondria staining by TMRE near the axotomy site (white dashed lines), whereas those depolarized mitochondria maintained GFP-Mito signals. MC, microgroove channels. (F and G) Representative images (F) and ratiometric kymograph (G) showing axotomy-induced ATP depletion. Cultured adult DRG neurons from 2-mo-old mice were transfected with red-shifted ATP probe GO-ATeam2, in which GFP and OFP were used as a FRET pair to monitor intracellular ATP levels with an affinity Kd of 2.3 mM at 37°C. Laser-based axotomy was applied (white bars in F, white arrow in G) along a distal axon. The green color is the cp173-mEGFP channel, and the red color is the OFP channel (mKOk). The ratiometric kymograph was generated by time-lapse imaging of a total of 100 frames with 5-s intervals. Axotomy was applied at the 51th frame (G). Note that axotomy triggers acute ATP depletion at millimolar levels in the vicinity of the injured site. Bars: (A–C) 10 µm; (D–G) 20 µm.

Axotomy depolarizes mitochondria in the vicinity of injured sites. (A) Representative images showing axonal mitochondria in adult DRG neurons before and after laser-based axotomy. Adult DRG neurons isolated from P60 mice were cotransfected with DsRed-Mito and GFP. Note that mitochondria at the axotomized site were immediately lost during axotomy (white arrows). (B and C) Kymographs showing axonal mitochondria labeled by DsRed-Mito (B) or colabeled by GFP-Mito and TMRE (C) before and after axotomy. The white arrows indicate laser-scanning sites (injured site), and the black arrows in the y axis show the laser execution time. Time-lapse images were captured to show that mitochondria in the vicinity suddenly shrunk (B) and lost the Δψm (TMRE staining; C). The images were first recorded at 5-s intervals for a total of 50 frames; the consecutive post-axotomy recording was collected at 5-s intervals for a total of 50 frames. (D and E) Representative images showing axotomy-induced depolarization of axonal mitochondria. Neurons were infected with pLenti-GFP (D) or GFP-Mito (E), and axons in the terminal chambers were loaded with 25 nM TMRE dye at DIV12, followed by laser-based axotomy and time-lapse imaging. Note that in neurons expressing GFP (D), axons were quickly broken up upon axotomy (white dashed lines), and a majority of mitochondria in the vicinity suddenly lost their TMRE staining (bottom right). In neurons expressing GFP-Mito (E), axotomy triggered a sudden loss of mitochondria staining by TMRE near the axotomy site (white dashed lines), whereas those depolarized mitochondria maintained GFP-Mito signals. MC, microgroove channels. (F and G) Representative images (F) and ratiometric kymograph (G) showing axotomy-induced ATP depletion. Cultured adult DRG neurons from 2-mo-old mice were transfected with red-shifted ATP probe GO-ATeam2, in which GFP and OFP were used as a FRET pair to monitor intracellular ATP levels with an affinity Kd of 2.3 mM at 37°C. Laser-based axotomy was applied (white bars in F, white arrow in G) along a distal axon. The green color is the cp173-mEGFP channel, and the red color is the OFP channel (mKOk). The ratiometric kymograph was generated by time-lapse imaging of a total of 100 frames with 5-s intervals. Axotomy was applied at the 51th frame (G). Note that axotomy triggers acute ATP depletion at millimolar levels in the vicinity of the injured site. Bars: (A–C) 10 µm; (D–G) 20 µm.

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