<p><b>A–C.</b> Real time imaging of mitochondrial movement within the axon of Hip neuron from PS1 mouse 7 days in culture. <b>A.</b> Phase image of the axon; cell body is in the upper right corner. <b>B.</b> Same axon with mitochondria visualized using TMRM. Scale bar, 10 µm. <b>C (a–c).</b> Recording of mitochondrial movement in live axon: arrow and circle indicate the progress of the same organelle along the axon with time. Images were acquired using LSM 510 laser scanning microscope (Carl Zeiss) with 100× oil DIC (1.4 na) lens. Scale bar, 5 µm. <b>D.</b> Mitochondria in PS1 neurons cover significantly shorter distances between stops in both, anterograde and retrograde directions compared to organelles in NTG, APP or APP/PS1 neurons. Almost no mitochondria in PS1 neurons cover distances longer than 10 µm. Number of organelles taken into analysis is the same as in (<b>F,G</b>). Blue – NTG; Red – APP; Orange – APP/PS1; Green – PS1. *p<0.001. <b>E.</b> Selective analysis of mitochondrial dynamics was done using analytical software (Analyze) that allows to trace each organelle from the first frame (<b>a</b>) through all 600 frames of the movie (stacked movie frames, <b>b</b>) to generate a final profile of movement (<b>c</b>). Resultant kymograph (<b>c</b>) is used to calculate velocities and identify the number of stationary and moving mitochondria. Rates of organelle motility in anterograde (<b>F</b>) and retrograde (<b>G</b>) directions include analysis of movement of 74 to 285 individual mitochondria in 24 to 33 neurons from at least three individual platings for each genotype. *, p<0.001; **, p<0.05. <b>H.</b> Length of 85–156 individual mitochondrion was estimated in five randomly selected axons in E17 neurons. *, p<0.001; **, p<0.0001. <b>I.</b> The number of organelles normalized per axonal length in embryonic neurons from NTG and AD mice used in axonal trafficking analysis. Number of mitochondria increases in APP and PS1 mice, and decreases in APP/PS1 cells comparing to NTG neurons. *, p<0.01. Colors as in <b>D</b>.</p
