00 ± 0.05 (at 12–13 DIV, 241 puncta) and 0.99 ± 0.04 (at 19–23 DIV, 263 puncta)]. These results suggest that EGFP-VAMP2 can be used as a marker of presynaptic sites and also

that their fluorescence intensity can be used as an estimate of the presynaptic total SV pool size. After the establishment of reliable markers for both axonal mitochondria and presynaptic sites, we designed live imaging analyses with different sampling frequencies and total imaging duration. The final goal of this study was to provide a comprehensive description of mitochondrial behavior in the axon. Individual mitochondria in the axon changed their state with time (Fig. 1A). Moving mitochondria showed frequent pauses, but most pauses were transient

and paused mitochondria restarted within seconds to minutes. A small fraction of mitochondria remained stationary for a prolonged period (over hours and see more days) and this transition from mobile to stationary state was important in the generation of a large population of stationary mitochondria in the axon. Therefore, the imaging experiments should provide data sufficient to determine the transition rates among moving mitochondria ([M]) and mitochondria in short pause ([SP]) and stationary state ([SS]) (Fig. 1B). An ideal imaging experiment monitors the entire process of state transitions of individual mitochondria with high sampling frequencies and long imaging durations. However, this is not practical with currently available fluorescence probes and the sensitivity of image detection devices because Pictilisib of photobleaching and phototoxicity. Instead, we first determined the rate of transition from stationary to mobile states by intermediate and low-frequency imaging (experimental design in Fig. 1C, actual data presented in Figs 3 and 4). Next, we measured the rate of mitochondria pauses Nintedanib (BIBF 1120) from time-lapse images at high frequency (experimental design in Fig. 1D, actual data presented in Figs 5-7). Finally, these quantitative measures were combined and the rate of transitions from short pause to stationary states was estimated (Fig. 8).

To analyse the stability [rate of transitions from stationary to mobile states ([SSM]); Fig. 1C] of axonal mitochondria on time scales of several hours, cultured hippocampal neurons expressing mCherry-OMP and EGFP-VAMP2 were imaged at intervals of 30 min for 3 h. Neurons at 12–13 DIV (2 weeks, 3482 mitochondria from n = 8 experiments) and 19–20 DIV (3 weeks, 4052 mitochondria from n = 7 experiments) were compared to examine the relationship between the maturity of neurons and stability of mitochondria (Fig. 3A and B). Fractions of synapses that contained mitochondria at t = 0 min were calculated (2 weeks, 43.2 ± 1.8%; 3 weeks, 56.9 ± 2.6%). Although the fraction was similar to previous studies (Shepherd & Harris, 1998; Chang et al.