Fig. 4: Differential stability of newly generated spines formed on callosal and non callosal neurons following traumatic brain injury.

a Scheme of the experimental design with retroAAV injected in the lesion area and timeline of the two-photon in vivo imaging. b Confocal images of retrogradely labeled callosal neurons in the cortex (Green: retroAAV-EGFP; red: NT435). Insets are four times magnified (top raw overlay; middle raw: retroAAV-EGFP; bottom raw: NT435).Scale bar equals 50 μm. c Representative timelapse series of an apical dendrite from a callosal neuron retrogradely-labeled with retroAAV-EGFP. Each image shows the same dendrite stretch at a specific experimental time point before and up to 42d post-injury (23 independent dentritic stretches and 8724 spines were followed). Cyan arrowheads indicate disappearing pre-existing spines. Gray arrowheads indicate disappearing newly-formed spines. Green dot indicate stable pre-existing spines. Magenta dots indicate stable newly-formed spines. B1: Baseline 1; B2: Baseline 2; Scale bar: 10 μm. d Scheme of the experimental design with the retroAAV injected in the spinal cord and timeline of the two-photon in vivo imaging. e Confocal images of retrogradely labeled CST neurons in the cortex (Green: retroAAV-EGFP; red: NT435). Insets are two times magnified (top raw overlay; middle raw:retroAAV-EGFP; bottom raw: NT435). Scale bar equals 50 μm. f Representative timelapse series of an apical dendrite from a CST neuron retrogradely-labeled with retroAAV-EGFP. Each image shows the same dendrite stretch at a specific experimental time point before and up to 42d post-injury (13 independent dentritic stretches and 4376 spines were followed). Cyan arrowheads indicate disappearing pre-existing spines. Gray arrowheads indicate disappearing newly-formed spines. Green dot indicate stable pre-existing spines. Magenta dots indicate stable newly-formed spines. B1: Baseline 1; B2: Baseline 2; Scale bar: 10 μm. g Quantifications comparing the persistence of all pre-existing spines (n = 377 spines analyzed) to all newly formed spines (n = 187 spines analyzed) (top panel) and the persistence of newly formed spines on callosal (n = 134 spines from nine dentritic stretches from four animals) and CST neurons (n = 55 spines analyzed from eight dendritic stretches from four animals) (bottom panel). All data (top and bottom) are analyzed using two-tailed Mann–Whitney test (p < 0.0001 in both cases) and presented as box plots (top panel: Pre-existing spines: minima:0, maxima:1,median = 0.8889; 25% percentile = 0.6667; 75% percentile = 1; newly formed spines: minima:0, maxima:1; median = 0.2111; 25% percentile = 0; 75% percentile = 0.7708 – bottom panel: CST: minima:0, maxima:1, median = 0; 25% percentile = 0; 75% percentile = 0.3333; Callosal: minima:0, maxima:1,median = 0.3333; 25% percentile = 0; 75% percentile = 1) Source data are provided as a Source Data file.