PDF neuropeptide signals independently of Bruchpilot-labelled active zones in daily remodelled terminals of Drosophila clock neurons

The small ventrolateral neurons (sLNvs) are key components of the central clock in the Drosophila brain. They signal via the neuropeptide Pigment-dispersing factor (PDF) to align the molecular clockwork of different central clock neurons and to modulate downstream circuits. The dorsal terminals of the sLNvs undergo daily morphological changes that have been shown to affect presynaptic sites organised by the active zone protein Bruchpilot (BRP), a homolog of mammalian ELKS proteins. Although the circadian plasticity of the sLNv terminals is well established, whether and how it is related to the rhythmic release of PDF remains ill-defined. Here, we combined expansion microscopy with labelling of active zones by endogenously tagged BRP to examine the spatial correlation between PDF-containing dense-core vesicles and BRP-labelled active zones. We found that the number of BRP-labelled punctae in the sLNv terminals remained stable while their density changed during circadian plasticity. The relative distance between BRP- and PDF-labelled punctae was increased in the morning, around the reported time of PDF release. Spontaneous dense-core vesicle release profiles of the sLNvs in a publicly available ssTEM dataset (FAFB) consistently lacked spatial correlation to BRP-organised active zones. RNAi-mediated downregulation of brp and other active zone proteins expressed by the sLNvs did not affect PDF-dependent locomotor rhythmicity. In contrast, down-regulation of genes of the canonical vesicle release machinery, the dense-core vesicle-related protein CADPS, as well as PDF impaired locomotor rhythmicity. Taken together, our study suggests that PDF release from the sLNvs is independent of BRP-organised active zones which seem not to be circadianly destroyed and re-established. ### Competing Interest Statement The authors have declared no competing interest.