Fig. 7: Blockade of gap junction-mediated bystander changes affects neuronal death and initiation of the regeneration process.

a The experimental setup to investigate the bystander effect from the whole spinal MN population by injecting a mixture of NB (green) and Dextran tracer (Dx, magenta) into the ventral root and axial muscles. b Whole-mount confocal image (from a selected stack of images close to pMN level) of the adult zebrafish spinal cord showing the traced MNs (NB⁺Dx⁺) and the dye-coupled neurons (NB⁺Dx^-). Arrowheads indicate the dye-coupled neurons (NB⁺Dx^-). c Quantification of the ratio between dye-coupled neurons (NB⁺Dx^-) to traced MNs (NB⁺Dx⁺) and the dye-coupled neurons (NB⁺Dx^-) soma size in different experimental conditions. d Whole-mount inverted confocal images show PI⁺ cells (appear black) and their spatial distribution at 3 dpi after administration of the gap-junction channel blocker carbenoxolone (CBX). e Single-plane confocal images show the incorporation of PI (magenta) from spinal neurons (HuC/D⁺; green) and their relative distance from the lesion site. White dashed lines indicate the PI⁺HuC/D⁺ neurons. f Quantification and analysis of the number of PI⁺HuC/D⁺ neurons and soma sizes in saline and CBX at 3 dpi. g Progression (as %) of spinal cord bridge formation during the regeneration process. h Quantification of regeneration progress at different time points after injury in saline- (control) or CBX-treated animals. i At 7 dpi, carbenoxolone (CBX) delays the initiation of the bridge formation. j Experimental setup and images showing the neuronal processes that cross the lesion site at 14 dpi and analysis of the number of detected neurites. Asterisks indicate the pMN cell bodies. CBX Carbenoxolone, CDF cumulative distribution (frequency), Dx Dextran tracer, NB neurobiotin (N-(2-aminoethyl) biotinamide hydrochloride), PI propidium iodide, SCI spinal cord injury. Data are presented as mean ± s.e.m. and as box plots showing the median with 25/75 percentile (box and line) and minimum–maximum (whiskers). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant. For detailed statistics, see Supplementary Table 1. Source data are provided as a Source Data file.