Fig. 3.

Presynaptic excitability at synapses onto axotomized neurons. a A representative neuron retrogradely labeled with a modified eGFP rabies virus via the axonal compartment. Enlarged region shows FM puncta colocalized with eGFP dendrites and spines (arrows). ImageJ ‘fire’ color look-up-table for FM puncta shown in the next panel. Scale bar, 20 µm. b Representative images show FM puncta colocalized with eGFP dendrites (outlined in white dashed lines) before and after field stimulation in uninjured control, and 24 h and 48 h post-axotomy. Arrows highlight destaining at spines. Scale bars, 10 µm. c FM unloading curves of colocalized puncta 24 h post-axotomy (control, n = 185 puncta; axotomy, n = 256 puncta) and 48 h post-axotomy (control, n = 232 puncta; axotomy, n = 322 puncta). Two-way ANOVA, Bonferroni post hoc test. Inset shows FM decay time constant (τ) for puncta with τ < 360 s (24 h control, n = 151; 24 h axotomy, n = 201; 48 h control, n = 211; 48 h axotomy, n = 304). b, c Unpaired two-tailed t-test. Each condition includes 5–6 chambers/neurons over 3 experiments. d Percent responsive and unresponsive FM puncta per neuron field (n = 8 fields/chambers; 4 experiments). Unpaired two-tailed t-test, axotomy vs. control for each time point. e Number of responsive and unresponsive FM puncta per frame at 48 h post-axotomy (n = 11 chambers; 5 experiments). Unpaired two-tailed t-test, for unresponsive puncta. f Representative mEPSC traces 48 h post-axotomy. g mEPSC frequency and amplitude at 48 h post-axotomy (control, n = 17 neurons; axotomy, n = 20 neurons; 4 experiments). Inset: cartoon depicts recordings from either uninjured control neurons (black) or directly injured neurons (red). h Analysis of mEPSC frequency and amplitude of cut neurons [cut (red), n = 10 neurons] compared to neighboring uncut neurons within axotomized chambers [uncut (gray), n = 10 neurons]. g, h Unpaired two-tailed t-test, Welch’s correction. i FM unloading of neighboring uncut neurons identified by lack of eGFP (uncut neighbors, n = 816 puncta), uninjured control neurons (uncut-labeled, n = 232), and axotomized labeled neurons (cut-labeled, n = 322). Two-way ANOVA, Bonferroni post hoc test; each condition, 5 chambers and 3 experiments. Decay time constant (τ) of FM puncta at 48 h post-axotomy (uncut-labeled, n = 211; cut-labeled, n = 304; and uncut neighbors unlabeled, n = 703). One-way ANOVA, Bonferroni post hoc test. *p < 0.05, ***p < 0.001. Error bars, s.e.m