Fig. 1

Axonal transport of autophagic vesicles in the optic nerve in vivo. A Schematic drawing of the experimental setup for two-photon real-time imaging of the optic nerve. In this experimental setup, the rat is maintained under deep anaesthesia, and its head is securely stabilized using a head holder. Throughout the surgical procedure, the rat is positioned on a heated pad to maintain its body temperature. Vital physiological parameters of the rat are closely monitored throughout the experiment. Using a transorbital approach, the optic nerve is carefully exposed and then subjected to in vivo imaging using a two-photon microscope. B Representative two-photon microscopy of the optic nerve. White arrows indicate the LC3 viral vector labeled autophagosomes in the single axon. C Representative kymographs of LC3 transport in the rat optic nerve. D–F Quantification of the number of moving (D), stationary (E) and moving divided into different transport directions (F) LC3 vesicles in the optic nerve of rats under physiological conditions. Error bars represent Mean ± SEM. G Quantification of proportions of different LC3 vesicles (anterograde, retrograde, and bidirectional/stationary vesicles) in the physiological situation. Error bars represent Mean ± SEM. H Quantification of the average velocity of LC3 vesicles in different directions of movement under physiological conditions. In all quantifications, a minimum of 10 axons per time point per animal was evaluated and a total of 8 animals were included. Figure A modified from biorender, https://app.biorender.com/