Figure 1.

Decrease of Ca_v2.2 accumulations and spontaneous Ca²⁺ transients in growth cones of trkBTK^−/− motoneurons corresponds to altered axon growth on laminin-221. (A) Representative images of axonal growth cones of wild type motoneurons cultured on laminin-221/211 for 5 days in vitro in the presence of BDNF and CNTF. TrkB receptors (green) and Ca_v2.2 calcium channels (magenta) were closely localized in growth cone protrusions, highlighted by white arrowheads (scale bar: 5 μm). (B) In trkBTK^−/− growth cones Ca_v2.2 accumulation (magenta) was affected in growth cone tips, whereas tau levels (green) were not altered (scale bar: 5 μm). Statistical analysis of Ca_v2.2 immunoreactivity in trkBTK^−/− axonal growth cones (0.54 ± 0.1, Q₂ 0.47, n = 4, N = 66) in comparison to wild type controls (1.00 ± 0.04, Q₂ 1.01, n = 6, N = 78) revealed a significant difference (p = 0.0015). Similar findings were obtained by normalizing Ca_v2.2 against the internal reference protein tau (trkBTK^+/+ 1.00 ± 0.08, Q₂ 0.95; trkBTK^−/− 0.65 ± 0.06, Q₂ 0.67; p = 0.0118) (C) The structural phenotype was accompanied by significant differences in the frequency of spontaneous calcium transients between control and trkBTK^−/− growth cones (Control 0.92 ± 0.13, Q₂ 0.60, N = 72; trkBTK^−/− 0.55 ± 0.09, Q₂ 0.30, N = 64; p = 0.0337). Representative recordings of control (upper trace, blue arrowheads) and trkBTK^−/− (lower trace, magenta arrowheads) growth cones showed a reduced number of calcium spikes when trkB signaling is impaired. (D) Wild type and trkBTK^−/− motoneurons were cultured on laminin-221/211 for 7 days in vitro in the presence of BDNF and CNTF and stained against tau (scale bar: 150 μm). TrkB mutant cells (316.4 ± 12.65 μm, Q₂ 314.5 μm, n = 7, N = 298) grew longer axons than controls (262.9 ± 11.79 μm, Q₂ 278.8 μm, n = 7, N = 283, p = 0.0093) (E) Treatment with 30 nM ω-conotoxin (CTX) caused enhanced axonal elongation in wild type motoneurons, but not in trkBTK^−/− cells.