Neurons with Multiple Axons Have Functional Axon Initial Segments

Multiple axons induced by pharmacological inhibition of GSK-3 activity in cultured primary hippocampal neurons have axon initial segments (AISs) and different AnkG distribution. A Representative images of neurons treated with DMSO (upper panel) or SB41 (lower panel) (scale bar, 50 μm). AISs were identified by AnkG antibodies (red), dendrites were identified by MAP2 antibodies (blue), and nuclei were stained by DAPI (white). Asterisks indicate neurons with multiple AISs. B, C Proportions of neurons with multiple AISs as indicated by AnkG (B, t 8 = 4.769, P = 0.0014) or neurofascin (C, t 4 = 5.253, P = 0.0063) antibodies in both groups (data from 3 (neurofascin) or 5 (AnkG) independent repeated experiments; **P <0.01, t-test). D Upper panel, representative image showing the measurement of AnkG on an AIS. The dashed line indicates the position of the AIS. Lower panel, the change of AnkG fluorescence intensity on an AIS along with distance. E Multiple-axon neurons had AISs closer to the soma. F The average length of AIS was shorter in multiple-axon neurons. G The fluorescence intensity of AnkG on each AIS was decreased in multiple-axon neurons. H The somatic AnkG level did not differ between single- and multiple-axon neurons. n = DMSO single 45, multiple 45; SB41 single 73, multiple 85 AIS, from 2 experiments; **P < 0.01 in DMSO or SB41 groups, ## P < 0.01 in single or multiple groups, two-way ANOVA