Fig 1: BDNF signaling is sufficient to induce Cav2.2 clustering and local Ca2+ transients at growth cone protrusions of embryonic motoneurons on laminin-221. (A) Representative images of axonal growth cones of non-pulsed and BDNF-pulsed axonal growth cones on laminin-221/211 stained against Cav2.2 (magenta) and APP (green) (scale bar: 5 µm). Upon BDNF pulse Cav2.2 channels clustered at growth cone tips as highlighted by white arrowheads. In the non-pulsed condition potential accumulations of Cav2.2 channels were rather detected in central growth cone areas as indicated by white circles. Cav2.2 levels revealed a significant increase upon BDNF pulse (no pulse 1.00 ± 0.04, Q2 1.00, n = 9, N = 221; 5' BDNF 1.93 ± 0.24, Q2 1.75, n = 9, N = 245; p = 0.0014). The ratio of Cav2.2 and APP immunoreactivities yielded similar results. (B) These structural changes upon treatment with BDNF were matched by significantly enhanced frequencies of spontaneous Ca2+ transients in comparison to non-pulse controls (no pulse 0.16 ± 0.08, Q2 0, IQR 0, N = 32; 5' BDNF 0.30 ± 0.08, Q2 0, IQR 2, N = 32; p = 0.0104). (Right panel) Identical growth cones were imaged prior to and 2 min after BDNF pulse. Representative recordings of non-pulsed and BDNF-pulsed traces showed increased numbers of Ca2+ spikes (magenta arrowheads) in response to BDNF. Upon BDNF the total number of calcium spikes almost doubled and the percentage of active growth cones displaying at least one spike per recording was greatly increased. (C) Representative images of non-pulsed and BDNF-pulsed trkBTK+/+ and trkBTK-/- growth cones stained against Cav2.2 (magenta) and synaptophysin (green) (scale bar: 5 µm). In wild type cells the acute application of BDNF resulted in increased Cav2.2 immunoreactivity at growth cone protrusions (indicated by white arrowheads). This effect was not visible in trkBTK-/- axonal growth cones, where Cav2.2 immunoreactivity rather occurred in central growth cone regions as emphasized by white circles. Synaptophysin appeared comparable in each condition serving as internal reference protein. (Right panel) Statistical significance was determined by the ratio of BDNF-pulsed vs. non-pulsed growth cones of trkBTK+/+ and trkBTK-/- motoneurons with respect to Cav2.2 alone (trkBTK+/+ 1.00 ± 0.06, Q2 1.00, n = 7, N(no pulse) = 88, N(5' BDNF) = 102; trkBTK-/- 0.75 ± 0.07, Q2 0.79, n = 7, N(no pulse) = 100, N(5' BDNF) = 106; p = 0.0184) and Cav2.2 signal intensities normalized against synaptophysin highlighting the reduced responsiveness of trkBTK-/- growth cones to acute BDNF application.