Microtubule disruption in primary neurons is an early response to oligomeric Aβ42. Primary cortical neurons were cultured as in Fig. , and after 12 days in vitro, were labeled for 30 min with 250 nM Tubulin Tracker, rinsed, and then exposed to 10 μM Aβ42 oligomers or vehicle. Live imaging on an Andor spinning disk confocal microscope began 30 min after addition of Aβ42. a Fluorescence and differential interference contrast (DIC) images were acquired every 5 min for 3 h. Neurite beading, microtubule accumulation in varicosities (arrowheads), and microtubule fragmentation (arrows) in neurites were present 3.5 h after Aβ42 addition, in the absence of significant cell death or BACE1 elevation []. In contrast, the morphology of vehicle-treated neurons was unaffected, with the exception of a moderate decrease of fluorescence intensity. DIC imaging revealed that neurites of Aβ42-treated neurons were intact and continuous, even though they exhibited beading and microtubule fragmentation, indicating that the observed microtubule disruption was not the result of physical degeneration of neurites. Insets show higher magnification images of the boxed region of the Aβ42-treated culture to accentuate varicosity formation and microtubule fragmentation. b The ratios of Tubulin Tracker fluorescence intensity (Int.) at 3.5 h (hours) to that at 0.5 h after Aβ42 treatment were calculated for 46 image fields from two separate experiments and averaged for Aβ42-treated and vehicle-treated neurons. Overall Tubulin Tracker fluorescence intensity decreased in neurons over time due to photo-bleaching, as indicated by fluorescence intensity ratios below 1. Nevertheless, the Tubulin Tracker fluorescence intensity ratio for Aβ42-treated neurons showed a small but highly significant decrease compared to vehicle treatment (p = 0.004), suggesting microtubule depolymerization and reduced stability of microtubule networks after only 3.5 h of Aβ42 exposure. Error bars SEM; **, p < 0.01. c To investigate longer Aβ42 treatment times and minimize photo-bleaching, primary neuron cultures were prepared as above and images collected once every 60 min for 6 h. Neurite beading, aberrant microtubule localization, and microtubule fragmentation were even more prevalent and pronounced in neurons after 6.5 h compared to 3.5 h of Aβ42 treatment, while vehicle-treated neurites continued to appear normal. Scale bars 20 μm for all images in a, c