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. 2017 Oct 30;10:346. doi: 10.3389/fnmol.2017.00346

Figure 2.

Figure 2

BDNF is necessary for Cav2.2 accumulation and local Ca2+ transients in growth cones of motor axons on laminin-221. (A) Images of axonal growth cones of wild type motoneurons cultured on laminin-221/211 for 5 days in vitro in the presence of BDNF, CNTF, or GDNF and stained against transmembrane protein APP (green) and Cav2.2 (magenta) (scale bar: 5 μm). With CNTF or GDNF Cav2.2 signals were significantly reduced in comparison to BDNF (BDNF 1.00 ± 0.11, Q2 1.00, n = 5, N = 130; CNTF 0.65 ± 0.05, Q2 0.65, n = 6, N = 133; GDNF 0.47 ± 0.04, Q2 0.47, n = 6, N = 145; p(B-C) = 0.0081, p(B-G) = 0.0002), particularly at axon terminal protrusions, whereas APP immunoreactivities were not decreased. Similar results were obtained by normalizing Cav2.2 intensities against internal reference protein APP. (B) In comparison to BDNF-treated motoneurons CNTF- and GDNF-treated cells displayed reduced frequencies of spontaneous Ca2+ transients in their corresponding growth cones (BDNF 0.89 ± 0.11, Q2 0.6, N = 75; CNTF 0.69 ± 0.13, Q2 0.3, N = 77; GDNF 0.50 ± 0.08, Q2 0.3, N = 73; p(B-C) = 0.0306, p(B-G) = 0.0078). (Right panel) Representative recordings of axonal growth cones of motoneurons cultured with BDNF (blue), CNTF (magenta) or GDNF (green) showing calcium spikes. (C) Motoneurons were cultured on laminin-221/211 for 7 days in vitro in the presence of BDNF, CNTF, or GDNF and stained against tau (scale bar: 150 μm). Both CNTF- (466.6 ± 35.41 μm, Q2 465.6 μm, n = 6, N = 263) and GDNF-treated (454.8 ± 19.28 μm, Q2 440.9 μm, n = 6, N = 200) motoneurons grew significantly longer axons (p(B-C) = 0.0256, p(B-G) = 0.0470) in comparison to BDNF-treated cells (356 ± 22.16 μm, Q2 342 μm, n = 6, N = 275).