12; unpaired t-test; Fig. 5A and B). Next, we asked whether the developmental changes and effects of TTX treatment on average velocities and short-pause rates were cargo specific. Membrane organelles positive for amyloid precursor protein (APP) are also known to be transported by kinesin-1, which mediates anterograde transport of axonal mitochondria (Kamal et al., 2000; Hirokawa et al., 2010). Therefore,
we compared the behavior of mCherry-OMP-positive mitochondria with that of APP-mCherry-positive membrane organelles. Cultured hippocampal neurons expressing APP-mCherry and EGFP-VAMP2 were imaged at intervals of 1 s for 10 min [2 weeks (12–13 DIV), n = 53 Antero, n = 32 Retro from seven cells; 3 weeks (19–20 DIV), n = 76 Antero, n = 48 Retro from eight cells; 3 weeks
(19–20 DIV) with TTX treatment, n = 78 Antero, n = 49 Retro from eight cells]. selleck products A short pause of APP-containing vesicles was defined as an event with inter-frame velocities < 0.25 μm/s and duration of more than 1 s, together with the occurrence of restart during observation periods. An average velocity was calculated using the same method as we used for mitochondria. Consistent with the previous work, APP-containing vesicles moved faster in the anterograde Anti-diabetic Compound Library in vivo direction than in the retrograde direction (Fig. 5C) (Kaether et al., 2000). The transport of APP-containing vesicles showed properties that were different from those of mitochondrial transport. Both the average velocities and short-pause rates of APP-containing vesicles were similar at 2 and 3 weeks after plating (average velocity: Antero, t127 = 1.14, P = 0.26; Retro, t78 = 1.34, P = 0.19; short-pause rate: Antero, t127 = 0.79, P = 0.43; Retro, t78 = 0.46, P = 0.65; unpaired t-test; Fig. 5C and D). In addition, TTX did not affect the transport of APP-containing vesicles (average velocity: Antero, t152 = 0.66, P = 0.51;
Retro, t95 = 0.09, P = 0.92; short-pause rate: DOK2 Antero, t152 = 0.28, P = 0.78; Retro, t95 = 0.34, P = 0.73; unpaired t-test; Fig. 5C and D). These results indicate that the regulation of organelle transport by neuronal maturation and activity is cargo specific. High-frequency time-lapse imaging revealed developmental regulation of mitochondrial transport in the axon (Fig. 5). In the presence of TTX, the short-pause rates of mobile mitochondria were reduced, suggesting the involvement of axonal excitability and associated events in the regulation of mitochondrial short pause. Many mitochondrial short pauses occurred near presynaptic sites [number of synaptic short pauses/number of all short pauses = 67 ± 6% (Antero) and 44 ± 5% (Retro); Fig. 6A]. However, even if mitochondrial short pause occurred randomly, short pauses near presynaptic sites could be observed by chance, due to the high density of presynaptic sites. To critically evaluate whether short pauses of mitochondria preferentially occur near presynaptic sites, experimental data were compared with values generated by a stochastic simulation.