Journal: Nature Communications
Article Title: Discovery of long-range inhibitory signaling to ensure single axon formation
Figure Lengend Snippet: NT-3 generated long-range Ca 2+ signaling from the axon to the cell body. a Long-range Ca 2+ wave. The relative change in the Cal-520 emission ratio (defined as R) was used as a measure of changes in Ca 2+ concentration. The pseudocolored images represent R after local application of PBS ( top ) or NT-3 ( bottom ) to the axon ( arrow ). Scale bars, 50 μm. b The mean amplitude of R treat / R 0 for 90 s during local application of NT-3 in the presence of the indicated inhibitors (PBS = 10, NT-3 = 16, xestospongin C = 9, ryanodine = 15, dantrolene = 18, SKF96365 = 12 neurons from three independent experiments). c , d The axon was exposed to NT-3 in the presence of the indicated inhibitors, and then minor neurite outgrowth (PBS = 21, NT-3 = 21, xestospongin C = 26, ryanodine = 25, dantrolene = 25, SKF96365 = 24 neurites from three independent experiments) c and axonal outgrowth (PBS = 7, NT-3 = 9, xestospongin C = 9, ryanodine = 9, dantrolene = 8, SKF96365 = 8 neurons from three independent experiments) d were measured. e Local application of NT-3 increased the quantity of phospho-CaMKI in the cell body. After local application of PBS ( top ) or NT-3 ( bottom ), hippocampal neurons were immunostained with antibodies against CaMKI ( green ) and phospho-Thr177 of CaMKI ( magenta ). The merged images ( right panels ) are shown. The graph plots the fluorescence intensities of total CaMKI ( green ) and CaMKI phosphorylated at Thr177 ( magenta ) and in the line. Scale bars, 20 μm. f NT-3-induced minor neurite retraction was abolished by Ca 2+ signaling inhibitors. The axon was exposed to NT-3 in the presence of the indicated inhibitors, and minor neurite outgrowth was measured (PBS = 27, NT-3 = 31, BAPTA = 47, STO-609 = 45, KN-93 = 40 neurites from three independent experiments). g , h Local application of indicated inhibitors to the axon. Minor neurite outgrowth (DMSO = 42, xestospongin C = 32, ryanodine = 37, dantrolene = 35, SKF96365 = 32 neurons from three independent experiments) g and axonal outgrowth (DMSO = 14, xestospongin C = 11, ryanodine = 13, dantrolene = 13, SKF96365 = 12 neurons from three independent experiments) h were measured. Error bars represent SEM. * P
Article Snippet: In some experiments, the following reagents were applied to the culture medium at least 30 min before the application of the NT-3 gradients as previouslydescribed : 2 μM xestospongin C, 20 μM dantrolene, 3 μM SKF96365, 100 μM ryanodine (Alomone Labs), 5 μM BAPTA-AM (Invitrogen), 1 μM KN-93 or 5 μM STO-609.
Techniques: Generated, Concentration Assay, Fluorescence