Fig. 8.

Spine density and presynaptic release properties of axotomized neurons treated with exogenous netrin-1. a Representative dendrites before and 48 h post-axotomy treated with vehicle (HBS) or netrin-1 (Ntn1) beginning at 40 h post-axotomy (inverted fluorescence). Arrows: new spines; red asterisks: eliminated spines. Scale bars, 10 µm. b Quantification of spine density illustrated in a. Axotomy: n = 33 dendrites; 7 neurons; #spines/TDL: 392/3696 µm (before), 263/3447 µm (after). Axotomy + netrin-1: n = 29 dendrites, 6 neurons; #spines/TDL: 293/3281 µm (before), 363/3417 µm (after). c Percent responsive FM puncta per neuron field at 48 h post-axotomy with HBS or netrin-1. n = 8–11 fields/chambers per condition over 5 experiments. d Number of responsive and unresponsive FM puncta from c. Significantly fewer unresponsive puncta followed axotomy compared to uninjured control (HBS). e, f Number of vGLUT1 and vGAT puncta per neuron area (uninjured control, axotomized + HBS, or axotomized + netrin-1) at 14 DIV. n = 8–9 neurons; 3 chambers per condition over 3 experiments. g Representative DCC immunostaining (turquoise) in uninjured control, post-axotomy, and post-axotomy + netrin-1 in cultures with similar spine densities. Neurons were retrogradely labeled with fluorescent protein (FP, magenta) using an mCherry modified rabies virus. Scale bar, 10 µm. h Quantification of DCC immunofluorescence per spine region-of-interest (ROI). ROI: 2 µm diameter circular region surrounding each spine. Control, n = 295 ROIs; axotomy, n = 293 ROIs; axotomy + Ntn1, n = 210 ROIs. 8 neuron fields/3 chambers per condition; 3 experiments. i Quantification of spine density following 24 h of control antibody (IgG ab.) or DCC function blocking antibody (DCC ab.). IgG: n = 33 dendrites; 8 neurons; #spines/TDL: 464/4864 µm (before), 419/4712 µm (after). DCC ab: n = 34 dendrites; 7 neurons; #spines/TDL: 404/4433 µm (before), 222/3647 µm (after). j Representative FP-labeled dendritic segments immunostained for vGAT (inverted) and vGLUT1 (inverted) following 24 h application of IgG or DCC antibodies (outlined dendrites, solid magenta line). Neurons were fixed at 15–16 DIV, older than the cultures in e, f. Scale bar, 10 µm. Quantification shown on the right. n = 23 neuron fields per condition; 3 chambers per condition over 3 experiments. b, i Repeated-measure two-way ANOVA, Bonferroni post hoc test; analyses included 1 chamber per condition for 3 experiments. c, j Unpaired two-tailed t-test. d–f, h One-way ANOVA, Bonferroni post hoc test. Error bars, s.e.m. *p < 0.05, ****p < 0.0001