Fig. 4

Optogenetic stimulation of cortical neurons after axotomy. A Cortical neurons (Tuj-1) express ChR2-eYFP after LV-ChR2 infection. White arrows in the high magnification image indicate neuritic expression of ChR2. Scale bar: 250 μm. B Optogenetic stimulation increased the expression of c-Fos in cortical neurons. Representative images of c-Fos (red) and ChR2 (green) immunostaining. Scale bar: 20 μm. C, D Optogenetic stimulation of cortical neurons 30 min after axotomy (C) resulted in reduced axon regeneration, while delivering the stimulation 6 h after axotomy (D) increased axon regeneration. Individual ChR2⁺ axon lengths were quantified (C: n = 16 images; D: n = 37–43 images). Data are expressed as mean ± s.e.m. ***p < 0.001 denotes significant differences in Student’s t-Test; **p < 0.01 denotes significant differences in Mann Whitney test. E Optical stimulation (470 nm light) of GFP-expressing cortical neurons did not induce any changes in axonal regeneration. Total growth area/microchannel was computed (n = 7–8 images). Data are expressed as mean ± s.e.m. F Representative images of GFP/YFP staining used to analyze axon regeneration when stimulation is applied 6 h after axotomy. Scale bar: 200 μm