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. 2021 Jan 21;8(1):ENEURO.0277-20.2020. doi: 10.1523/ENEURO.0277-20.2020

Figure 4.

Figure 4.

Trk receptor inhibition protects axons from NGF deprivation but not from BDNF deprivation, whereas p75NTR deficiency confers no protection to axons established in NGF or BDNF. A, Protein lysates collected from E13.5 DRG explants cultured in the presence of NGF (12.5 ng/ml) or BDNF (37.5 ng/ml) for 48 h were analyzed by immunoblot against TrkA, TrkB, TrkC, p75NTR, and βIII-tubulin. B, p75NTR knock-out does not rescue axons from degeneration after NGF or BDNF withdrawal. DRG explants from mixed-genotyped E13.5 litters were cultured in the presence of NGF or BDNF for 48 h and then either maintained or withdrawal from trophic support for 24 h before being lived stained with Calcein-AM. NGF scale bar: 1000 μm; BDNF scale bar: 500 μm. C, Quantification of Calcein-AM intensity normalized by axonal area and relative to wild-type control. Non-significant difference was observed between wild-type and p75NTR-null DRG explants deprived of NGF or BDNF (n = 4 embryos in NGF and n = 4 embryos in BDNF from pooled litters). Analyzed by two-way ANOVA and Tukey’s post hoc comparison and plotted with median and SEM; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. D, DRG explants cultured in NGF or BDNF were either maintained in trophic media or withdrawn from trophic support with or without the Trk inhibitor K252a (200 nm) for 24 h before fixing, immunostaining for βIII-tubulin, and imaged by epifluorescence microscopy. NGF scale bar: 1000 μm; BDNF scale bar: 500 μm. E, K252a rescued degeneration induced by NGF deprivation but not by BDNF deprivation. Quantification of axonal area as a function of the distance from the soma using Axoquant 2.0 and plotted in 500-μm binned segments relative to 0–500 μm NGF control (upper panel) or BDNF control (lower panel). The relative axonal area was analyzed by two-factor ANOVA followed by Tukey’s post hoc comparison and plotted with mean and SEM (n = 3 embryos per condition for each condition; data shown are representative of three independent experiments); *ctr. versus dep. 24 h; #dep. 24 h versus dep. 24 h + K252a; ns: non-significant, **p < 0.01, ***p < 0.001, ****p < 0.0001.