(A) Schematic of retrograde labeling of corticospinal neurons. (B) Representative fluorescence images of corticospinal neurons identified by retrograde labeling after Fluoro-Gold injections into the cervical spinal cords of adult GFP-M mice. Sagittal sections of the mouse brain were immunostained with α2δ2 antibody (n = 4 independent replicates). Scale bar: 50 μm. (C) Immunoblot shows α2δ2 expression in the mouse sensory-motor cortex during postnatal development. Under reducing conditions, the α2δ2 antibody recognizes 2 bands at approximately 130 and 105 kDa. Tuj1 is used as loading control. (D) Quantification of C. Data normalized using loading control (linear trend test **P < 0.01, n = 3 biological replicates). (E) Representative fluorescence images of corticospinal neurons from mouse brains at different ages. Scale bar: 50 μm. (F) Quantification of E. Box plot (minimum to maximum) and line at median (1-way ANOVA followed by Dunnett post test *P < 0.05; **P < 0.01; P7 n = 5, P14 n = 3, and P28 n = 3 mice, 60–95 neurons per condition). (G) Raster plots show spontaneous firing within layer V of the sensory-motor cortex at different stages of brain development. (H) Quantification of G. Mean and SEM (linear trend test *P < 0.05; P7 n = 5, P14 n = 7, and P28 n = 8 mice). (I) Schematic representation of C5 SCI experimental model. (J) Representative fluorescence images of retrogradely labeled corticospinal neurons (yellow arrows) 7 days after C5 SCI. DPO, days after operation. Sagittal sections of the mouse brain (right hemisphere) were immunostained with α2δ2 antibody. Scale bar: 50 μm. (K) Quantification of J. Mean and SEM (unpaired 2-tailed Student’s t test **P < 0.01; sham n = 4 and SCI n = 4 mice, 229–302 neurons per condition).