Figure S4.
Functions of stress-induced axonal responses and validation of the effects of several compounds on such responses. (A) Representative images of the straightened axon before and after the flux (top) and automatic detection of the axon beading (bottom). Bar = 20 μm. (B) Paired comparison of axon bead density in soma chamber (soma) and central injury chamber (fluxed) (N = 6, 6, 21, 15). (C) Representative images showing the [Ca2+]axon intensity in straightened axons before and after the flux. Bar = 20 μm. (D) Rat hippocampal neurons co-expressing Lifeact-RFP and GCaMP-6f were fluxed at 50 μl/min. Representative field showing deformation of less-stressed axonal segments near the boundary of the AoC device (1#, boundary) or the more severely stressed distal axonal part (2#, central), with the beads indicated by white arrowheads. Bar = 30 μm (left) and 10 μm (right). (E) Quantification of D (N = 10, 12). (F) Axons expressing Lifeact-GFP were pretreated with EDTA (0.5 mM), BAPTA-AM (10 μM), and PNB (50 μM) for 30 min, then exposed to 50 μl/min flux for 180 s. Left panels display representative time-lapse images illustrating the axon deformation before and during flux; right panels show the automatically detected beads highlighted in magenta. Bar = 5 μm. (G) Curves depict fluctuations in axon bead number in F. (H) Peak bead number quantification in F (N = 41, 20, 28, 18). (I) Quantification of [Ca2+]axon wave frequency along the same axon before and during 50 μl/min flux (N = 58). (J) Representative images showing the resting [Ca2+]axon intensity before the flux in control, PNB (50 μΜ), and ML-7 (10 μΜ) treated axons. Bar = 20 μm. (K) Quantification of J (N = 64, 40, 49). (L) Quantification of the peak values of the ΔF/F0 [Ca2+]axon changes, related to Fig. 6 C (N = 26, 42). Results are shown as mean ± SEM; in B, E, and I, paired two-tailed Student’s t test; in H, K, and L, unpaired two-tailed Student’s t test; **P < 0.01, ***P < 0.001, n.s., non-significant.

Functions of stress-induced axonal responses and validation of the effects of several compounds on such responses. (A) Representative images of the straightened axon before and after the flux (top) and automatic detection of the axon beading (bottom). Bar = 20 μm. (B) Paired comparison of axon bead density in soma chamber (soma) and central injury chamber (fluxed) (N = 6, 6, 21, 15). (C) Representative images showing the [Ca2+]axon intensity in straightened axons before and after the flux. Bar = 20 μm. (D) Rat hippocampal neurons co-expressing Lifeact-RFP and GCaMP-6f were fluxed at 50 μl/min. Representative field showing deformation of less-stressed axonal segments near the boundary of the AoC device (1#, boundary) or the more severely stressed distal axonal part (2#, central), with the beads indicated by white arrowheads. Bar = 30 μm (left) and 10 μm (right). (E) Quantification of D (N = 10, 12). (F) Axons expressing Lifeact-GFP were pretreated with EDTA (0.5 mM), BAPTA-AM (10 μM), and PNB (50 μM) for 30 min, then exposed to 50 μl/min flux for 180 s. Left panels display representative time-lapse images illustrating the axon deformation before and during flux; right panels show the automatically detected beads highlighted in magenta. Bar = 5 μm. (G) Curves depict fluctuations in axon bead number in F. (H) Peak bead number quantification in F (N = 41, 20, 28, 18). (I) Quantification of [Ca2+]axon wave frequency along the same axon before and during 50 μl/min flux (N = 58). (J) Representative images showing the resting [Ca2+]axon intensity before the flux in control, PNB (50 μΜ), and ML-7 (10 μΜ) treated axons. Bar = 20 μm. (K) Quantification of J (N = 64, 40, 49). (L) Quantification of the peak values of the ΔF/F0 [Ca2+]axon changes, related to Fig. 6 C (N = 26, 42). Results are shown as mean ± SEM; in B, E, and I, paired two-tailed Student’s t test; in H, K, and L, unpaired two-tailed Student’s t test; **P < 0.01, ***P < 0.001, n.s., non-significant.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal