Robo2 regulates synaptic oxytocin content by affecting actin dynamics

• Published in: eLife Vol. 8
• Main Authors: Anbalagan, Savani, Blechman, Janna, Gliksberg, Michael, Gordon, Ludmila, Rotkopf, Ron, Dadosh, Tali, Shimoni, Eyal, Levkowitz, Gil
• Format: Journal Article
• Language: English
• Published: England eLife Science Publications, Ltd 10-06-2019; eLife Sciences Publications Ltd; eLife Sciences Publications, Ltd
• Subjects: Actin; Actins - metabolism; Animal genetic engineering; Animals; Animals, Genetically Modified; arginine-vasopressin; cdc42 GTP-Binding Protein - genetics; cdc42 GTP-Binding Protein - metabolism; Cdc42 protein; Cofilin; Developmental Biology; Displays (Marketing); Filaments; Fishes; Fluorescence Recovery After Photobleaching; Genetic engineering; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; Homeostasis; hypothalamus; Intracellular Signaling Peptides and Proteins - genetics; Intracellular Signaling Peptides and Proteins - metabolism; Microscopy, Fluorescence, Multiphoton; Muscle proteins; Mutation; neuroendocrine; neurohypophysis; Neuropeptides; Neurophysiology; Neuroscience; Novels; Oxytocin; Oxytocin - genetics; Oxytocin - metabolism; Physiological aspects; Physiology; Potassium; Proteins; Receptors, Immunologic - genetics; Receptors, Immunologic - metabolism; Science; Signal Transduction - genetics; synapse; Synapses - metabolism; Transmission electron microscopy; Vesicles; Zebrafish; Zebrafish - genetics; Zebrafish - metabolism; Zebrafish Proteins - genetics; Zebrafish Proteins - metabolism; Germany; United States > US; Israel; neurohypophysis; oxytocin; arginine-vasopressin; neuroendocrine; developmental biology; neuroscience; synapse; zebrafish; hypothalamus
• ISSN: 2050-084X; 2050-084X
• DOI: 10.7554/eLife.45650

The regulation of neuropeptide level at the site of release is essential for proper neurophysiological functions. We focused on a prominent neuropeptide, oxytocin (OXT) in 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 enriched with polymerized actin. Perturbation of actin filaments by either cytochalasin-D or conditional Cofilin expression resulted in decreased synaptic OXT levels. Genetic loss of
or
displayed decreased synaptic OXT content and
mutants displayed reduced mobility of the actin probe Lifeact-EGFP in OXT synapses. Using a novel transgenic reporter allowing real-time monitoring of OXT-loaded vesicles, we show that
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
mutants. Our results link Slit3-Robo2-Cdc42, which controls local actin dynamics, with the maintenance of synaptic neuropeptide levels.