Eichel-Vogel, M. A.; Soong, D.; Sequeira, M. N.; Marshall-Phelps, K. L.; Bin, J. M.; Lyons, D. A.

Axon diameter varies up to 100-fold between distinct neurons in the central nervous system with larger axons exhibiting proportionally faster conduction velocity. Axon diameter influences myelination, can be dynamically regulated, which might help fine-tune neural circuit function, and is altered in several diseases. Despite its importance, mechanisms regulating axon diameter remain poorly understood. This gap in understanding is due in part to the limitations of fixed tissue analyses, such as electron microscopy, which cannot be scaled up to execute discovery screens. To address this, we developed a high-resolution, high-content imaging-based in vivo platform to identify pharmacological modulators of axon diameter in zebrafish. We focused on the Mauthner neuron, whose axon diameter growth can be monitored during development. To facilitate our high-content chemical screen, we developed an automated high-resolution imaging and image analysis pipeline to assess changes in Mauthner axon diameter in transgenic reporter animals. We screened 880 compounds and identified 33 that altered Mauthner axon diameter. Validating this discovery pipeline, we confirmed that compounds that affect beta-2 adrenoceptor and dopamine signaling increase axon diameter in separate follow-up studies. This represents the first discovery screen for axon diameter regulators, providing novel entry points to study the biology of axon diameter regulation.