Robo2 regulates synaptic oxytocin content by affecting actin state

The regulation of neuropeptide level at the site of release is essential for proper neurophysiological functions. We focused on a prominent neuropeptide, oxytocin (OXT), and used the zebrafish as an in vivo model to visualize and quantify OXT content at the resolution of a single synapse. We found that OXT-loaded synapses were surrounded by polymerized actin. Perturbation of actin filaments by cytochalasin-D resulted in decreased synaptic OXT levels. Live imaging of the actin probe, Lifeact-EGFP, showed reduced mobility in OXT synapses in robo2 mutants, which displayed decreased synaptic OXT content. Using a novel transgenic reporter line allowing real-time monitoring of OXT-loaded vesicles, we showed that robo2 mutants display slower rate of vesicles accumulation. OXT-specific expression of dominant-negative Cdc42, which is a key regulator of actin dynamics and a downstream effector of Robo2, led to a dose-dependent increase in OXT content in WT, and a dampened effect in robo2 mutants. Our results link Robo2-Cdc42 signalling, which controls local actin dynamics, with the maintenance of synaptic neuropeptide levels.