Fig. 5

Absence of inhibitory terminals following distal axotomy and frequency of their spontaneous release events. a Representative images of fixable FM4-64FX puncta (red) and vGLUT1 (green) co-immunolabeling in uninjured chambers and 48 h following axotomy. White circles highlight vGLUT1 expression at FM-labeled terminals. Scale bars, 10 µm. Fraction of vGLUT1 + FM puncta per neuron field at 48 h post-axotomy normalized to uninjured controls. n = 18 neuron fields; 5 chambers over 3 experiments. b Fixable FM4-64FX puncta (red) and GAD67 (green) co-immunolabeling. Quantification of GAD67-positive FM puncta at 48 h post-axotomy normalized to control. n = 21 neuron fields; 5 chambers over 3 experiments. c Number of vGLUT1 and vGAT puncta per neuron area (axotomized or control) 48 h post-axotomy. n = 8–9 neurons; 3 chambers per condition over 3 experiments. a–c Unpaired two-tailed t-test. d Representative traces of mIPSC recordings 48 h post-axotomy. e Quantification of mIPSC frequency and amplitude 48 h post-axotomy (control, n = 9 neurons; axotomy, n = 17 neurons). (mIPSC frequency: unpaired two-tailed t-test with Welch’s correction, p = 0.05; mIPSC amplitude: unpaired two-tailed t-test, p = 0.62). f Analysis of mIPSC frequency and amplitude from axotomized devices previously shown in e comparing neurons with axons that extended into the axonal compartment and were cut (cut, n = 8 neurons), to neurons that did not extend axons into the compartment and were not cut (uncut, n = 9 neurons). (mIPSC frequency: unpaired two-tailed t-test, p = 0.94; mIPSC amplitude: unpaired two-tailed t-test, p = 0.51). d–f Data shown were combined from 3 chambers. g Representative dendritic segments (retrogradely labeled with eGFP rabies virus) showing spines that are labeled with vGLUT1 (red) or vGAT (magenta) antibodies. White open circles highlight dendritic spines with vGLUT1 and/or vGAT synapses. h Fraction of vGLUT1- and vGAT-positive spines at 24 h post-axotomy. n = 12–14 neuron fields; 7 neurons per condition; #spines: 458 (uninj. cntl), 394 (axot.); 3 chambers over 3 experiments. i Fraction of vGLUT1- and vGAT- positive spines at 48 h post-axotomy. n = 11–15 neuron fields; 8–9 neurons per condition; #spines: 460 (uninj. cntl), 413 (axot.); 3 chambers over 3 experiments. Scale bars, 5 µm. Unpaired two-tailed t-test. **p < 0.01, ***p < 0.001. Error bars, s.e.m