Fig. 6
Influence of local activity and transcription on the initiation of injury-induced synaptic remodeling. a Representative images of neurons within microfluidic chambers retrograde labeled with eGFP rabies virus (inverted grayscale) before and 24 h post-axotomy with vehicle or local activity blockade solution (ABS) applied to axons for 1 h during axotomy. Inset shows zoomed in dendritic regions. Image and inset scale bars, 50 and 5 µm, respectively. b Quantification of before and after spine density data described in a. Axotomy (vehicle): n = 29 dendrites; 5 neurons; 3 chambers over 3 experiments; #spines/TDL: 437/4185 µm (before), 274/3868 µm (after). Axot. (ABS): n = 33 dendrites; 5 neurons; 3 chambers over 3 experiments; #spines/TDL: 426/3889 µm (before), 446/3700 µm (after). c Quantification of spine density changes following application of transcription blocker (DRB) to the somatodendritic compartment for 1 h during axotomy within microfluidic chambers. DRB (uninj.): n = 27 dendrites; 6 neurons; 3 chambers over 3 experiments; #spines/TDL: 362/4186 µm (0 h), 262/3934 µm (24 h after). DRB (axot.): n = 28; 6 neurons; 3 chambers over 3 experiments; #spines/TDL: 343/3414 µm (before) 283/3248 µm (after). b, c Unpaired two-tailed t-test. d Percentage of responsive and unresponsive FM puncta at 48 h post-axotomy. n = 6 neuron fields/chambers per condition over 3 experiments. Unpaired two-tailed t-test for % responsive. e FM 5–95 unloading following 1 h application of DRB during axotomy/control. Uninj. DRB: n = 1580 puncta; 6 chambers per condition over 3 experiments. Axot. DRB: 2213 puncta; 6 chambers per condition over 3 experiments. f Percent of responsive and unresponsive puncta at 48 h post-axotomy following application of TTX to the somatodendritic compartment for 1 h during injury. n = 6 neuron fields/chambers per condition over 3 experiments. Unpaired two-tailed t-test, % responsive. g FM unloading curves following application of TTX. Uninj. TTX: n = 1360 puncta; 6 chambers per condition over 3 experiments. Axot. TTX: n = 1648 puncta; 6 chambers per condition over 3 experiments. Two-way ANOVA, Bonferroni post hoc test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Error bars, s.e.m
