In Vivo Fast Nonlinear Microscopy Reveals Impairment of Fast Axonal Transport Induced by Molecular Motor Imbalances in the Brain of Zebrafish Larvae

Abstract

Cargo transport by molecular motors along microtubules is essential for the function of eukaryotic cells, in particular neurons in which axonal transport defects constitute the early pathological features of neurodegenerative diseases. Mainly studied in motor and sensory neurons, axonal transport is still difficult to characterize in neurons of the brain in absence of appropriate in vivo tools. Here, we measured fast axonal transport by tracing the second harmonic generation (SHG) signal of potassium titanyl phosphate (KTP) nanocrystals (nanoKTP) endocytosed by brain neurons of zebrafish (Zf) larvae. Thanks to the optical translucency of Zf larvae and to the perfect photostability of nanoKTP SHG, we achieved a high scanning speed of 20 frames (of ≈90 μm × 60 μm size) per second in Zf brain. We focused our study on endolysosomal vesicle transport in axons of known polarization, separately analyzing kinesin and dynein motor-driven displacements. To validate our assay, we used either loss-of-function mutations of dynein or kinesin 1 or the dynein inhibitor dynapyrazole and quantified several transport parameters. We successfully demonstrated that dynapyrazole reduces the nanoKTP mobile fraction and retrograde run length consistently, while the retrograde run length increased in kinesin 1 mutants. Taking advantage of nanoKTP SHG directional emission, we also quantified fluctuations of vesicle orientation. Thus, by combining endocytosis of nanocrystals having a nonlinear response, fast two-photon microscopy, and high-throughput analysis, we are able to finely monitor fast axonal transport in vivo in the brain of a vertebrate and reveal subtle axonal transport alterations. The high spatiotemporal resolution achieved in our model may be relevant to precisely investigate axonal transport impairment associated with disease models.

3D Real-Time Two-Photon Microscopy Device for Single-Particle Holographic Tracking (3D-Red Shot)

“…During the experiment, that last several hours, we did not observe any alteration of the morphology of the cells, consistently with the extremely low absorption of the NIR excitation laser both by the cells and by the NC themselves. Thanks to its two-photon operation, the setup has the potential to be adapted to the observation of NC transport in more complex tissues as for instance in zebrafish larvae, in a superlocalization regime and at a much faster rate than commercial fast point scanning two-photon microscopy setups when dealing with 3D imaging …”

“…Thanks to its two-photon operation, the setup has the potential to be adapted to the observation of NC transport in more complex tissues as for instance in zebrafish larvae, in a superlocalization regime and at a much faster rate than commercial fast point scanning two-photon microscopy setups when dealing with 3D imaging. 34…”