Figure 2

Injury-induced KCC2 downregulation in spinal cord motoneurons is prevented in CaMKII-KCC2 mice. (A,B) Representative transverse L4-L5 spinal cord sections visualizing KCC2 expression in motoneurons at 3-days post-SNC, from wild-type (A: a–c), and CaMKII-KCC2 (B: a–c), mice. Immunofluorescence for KCC2 (green), ChAT (red) and DAPI (blue). (A: a,B: a) Scale bar, 500 μm. At low magnification, red motoneuron somas are clearly localized to Rexed laminae IX in both the injured-side (inj) and uninjured-side (non) ventral horns. (A: b,c,B: b,c) Scale bar, 20 μm. At high magnification, plasmalemmal KCC2 immunofluorescence in motoneurons is easily visible as the green border along the red soma perimeter. For wild-type mice, plasmalemmal KCC2 immunofluorescence is strong in uninjured-side motoneurons (A: b), but weak in injured-side motoneurons (A: c). For CaMKII-KCC2 mice, plasmalemmal KCC2 immunofluorescence is strong in both uninjured-side (B: b), and injured-side (B: c), motoneurons. (C–E) Truncated violin plots quantifying plasmalemmal KCC2 immunofluorescence ([plasmalemmal KCC2 intensity]/[soma pixel perimeter]), for individual motoneuron somas from injured-side (inj) and uninjured-side (non) ventral horns (L4-L5, Rexed lamina IX), for wild-type (WT-inj, WT-non), and CaMKII-KCC2 (KCC2-inj, KCC2-non), mice before or at various time-points after SNC. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; Kruskal–Wallis test, post-hoc Dunn’s multiple comparisons test. Cohorts [WT-inj, WT-non, KCC2-inj, KCC2-non]. (C) Before SNC, n = [32, 35, 34, 35] cells; 3 wild-type, 3 CaMKII-KCC2 mice. (D) 3-days post-SNC, n = [44, 38, 27, 27] cells; 3 wild-type, 2 CaMKII-KCC2 mice. (E) 42-days post-SNC, n = [33, 33, 27, 33] cells; 3 wild-type, 3 CaMKII-KCC2 mice.