To combine these two separate experimental data, event frequencies should be normalised by the unit length of the axon (axonal short-pause rates, axonal appearance and disappearance rates; see ‘Materials and methods’; Fig. 8). The axonal appearance and disappearance rates were measured from the same experimental

data shown in Fig. 3 (Fig. 1C). The short-pause rate of individual mitochondria was suppressed by TTX treatment at 3 weeks (Fig. 5B). However, the axonal short-pause rate was not changed by TTX treatment because the number of mobile mitochondria was increased by TTX treatment (Figs 3I and 8). By using these normalised rates, we could calculate the stabilisation rates at different conditions ([SPSS]; Fig. 8). The stabilisation rate Fluorouracil mouse near synapses ([SPSS]synaptic) declined significantly from 2 to 3 weeks (1.01 vs. 0.53%) and was modulated by TTX treatment. Because stabilisation rates away from synapses ([SPSS]non-synaptic) were less affected by culture periods and TTX treatment, regulation of the stabilisation rate near synapses is likely Obeticholic Acid ic50 to be the parameter that is important for the control of mitochondrial replacement along the axon. Although the axonal appearance rate of

mitochondria near synapses ([MSS]synaptic) was more than twofold higher at 2 weeks, this increase was counterbalanced by the comparable rate of disappearance ([SSM]synaptic). It is likely that there exists a mechanism that keeps the balance between [MSS] and [SSM], as these rates were maintained in parallel in all experimental conditions (Fig. 8). This regulation may be important to keep the density of both synaptic and non-synaptic mitochondria constant with time. We report here the dynamic properties of axonal mitochondria using live-cell imaging with multiple sampling frequencies ranging from seconds to days. High-frequency image sampling is necessary to trace the accurate positions of mobile mitochondria, transported by motor proteins with their velocity of 0.1–1.4 μm/s (De Vos & Sheetz, 2007; MacAskill & Kittler, 2010).

In turn, the probability of transitions between stationary and mobile states is low (a few events per hour within an image area; Fig. 8) and time-lapse imaging with longer durations is required. Here we performed time-lapse imaging with high (intervals of 3 s), intermediate (intervals of 30 min) O-methylated flavonoid and low (intervals of 1 day) frequencies. Our results demonstrated that mitochondrial dynamics on multiple time scales differ between developmental stages and are regulated by neuronal activity and proximity to synaptic sites. To understand the dynamics of axonal mitochondrial distribution, mitochondrial properties in mobile and stationary states, and the transition process between them should be examined (Fig. 1). Our analyses revealed that the properties of stationary mitochondria are highly regulated by neuronal maturation and activity.