The majority of axonal mitochondria were stationary for 3 h at both developmental stages, with a small number of appearance (red arrowheads) and disappearance (white arrowheads) events. The mitochondrial population identified at t = 0 min buy Quizartinib progressively changed their positions with time. The fraction of mitochondria that remained at their initial positions was calculated as a position survival rate

P(t) (see ‘Materials and methods’). To examine the relationship between the proximity to presynaptic sites and mitochondrial dynamics, P(t) was measured from mitochondria near presynaptic sites (synaptic) and also away from presynaptic sites (non-synaptic; Fig. 3D and E). Because mitochondria found at t = 0 min included both stationary and mobile mitochondria, Δ(P(0) − P(180)) was not

an appropriate estimate of mitochondria that started to move during the 180 min observation period. Natural Product Library Instead, we used Δ(P(30) − P(180)) as an index of the transition from stationary to mobile state (Fig. 3G and H). Using this index, we found that synaptic mitochondria were less likely to restart translocation than non-synaptic mitochondria at both developmental stages (2 weeks, t14 = 4.32, P < 0.001; 3 weeks, t12 = 3.57, P = 0.004; unpaired t-test; Fig. 3H). Both synaptic and non-synaptic mitochondria were less likely to transit to mobile state at 3 weeks than at 2 weeks (all, t13 = 9.65, P < 0.001; synaptic, t13 = 8.05, P < 0.001; non-synaptic, t13 = 4.89, P < 0.001; unpaired t-test; Fig. 3H). The treatment of neurons at 20 DIV with the sodium channel blocker TTX increased the transition probability to mobile state (3 weeks + TTX, 3297 mitochondria from n = 7 experiments; Cediranib (AZD2171) all, t12 = 4.72, P < 0.001; unpaired t-test; Fig. 3C,E,F and H). This effect was present in both synaptic and non-synaptic mitochondria (synaptic, t12 = 3.95, P = 0.002; non-synaptic, t12 = 3.88, P = 0.002; unpaired t-test; Fig. 3H). These results suggest that neuronal maturation, proximity

to synaptic sites and neuronal activity affect the stability of stationary mitochondria in the axon. We estimated the fraction of mobile mitochondria at t = 0 min [mobile fraction; calculated from P(t) at t = 0, 30 and 60 min; see Eqn (3) in 'Materials and methods'] (Fig. 3G). The mobile fraction at 3 weeks was smaller than at 2 weeks (t13 = 4.98, P < 0.001; unpaired t-test; Fig. 3I) and at 3 weeks with TTX (t12 = 3.82, P = 0.002; unpaired t-test; Fig. 3I). These results suggest that the ratio of mobile to stationary mitochondria in the axon was dependent on neuronal maturation and activity. In time-lapse imaging over 3 h, the majority of axonal mitochondria imaged at the initial time point remained stationary throughout the experiments (Fig. 3D–F), suggesting that the duration of stationary state is usually longer than several hours.