Widespread Receptivity to Neuropeptide PDF throughout the Neuronal Circadian Clock Network of Drosophila Revealed by Real-Time Cyclic AMP Imaging

Abstract

The neuropeptide PDF is released by sixteen clock neurons in Drosophila and helps maintain circadian activity rhythms by coordinating a network of approximately 150 neuronal clocks. Whether PDF acts directly on elements of this neural network remains unknown. We address this question by adapting Epac1-camps, a genetically encoded cAMP FRET sensor, for use in the living brain. We find that a subset of the PDF-expressing neurons respond to PDF with long-lasting cAMP increases and confirm that such responses require the PDF receptor. In contrast, an unrelated Drosophila neuropeptide, DH31, stimulates large cAMP increases in all PDF-expressing clock neurons. Thus, the network of approximately 150 clock neurons displays widespread, though not uniform, PDF receptivity. This work introduces a sensitive means of measuring cAMP changes in a living brain with subcellular resolution. Specifically, it experimentally confirms the longstanding hypothesis that PDF is a direct modulator of most neurons in the Drosophila clock network.

PDF and cAMP enhance PER stability in Drosophila clock neurons

“…The positive effect of PDF on cAMP levels in PDFR-containing target neurons (12) suggests that an increase in cAMP levels might enhances PER stability. To test this possibility, we turned to the PER degradation S2 cell assay recently described.…”

“…This assay was combined with the addition of forskolin (FSK) or the addition of the cAMP analog Sp-adenosine-3′,5′-cyclic monophosphorothioate triethylamine (Sp-cAMPS). FSK activates adenylyl cyclase and has been previously used to phenocopy the effects of PDF in brain-imaging experiments (12). Sp-cAMPS is a specific activator of cAMP-dependent protein kinases (19).…”

“…Because SpcAMP may exaggerate physiological levels of cAMP induced by PDF, the assay was repeated but with the application of PDF rather than Sp-cAMPS directly onto the dissected fly brains. The peptide concentration was 10 μM, which is sufficient to induce the cAMP response within circadian neurons (12).…”

“…It is expressed in a broad subset of clock neurons, and a pdfr deletion is almost identical to the pdf 01 phenotype, including the advanced evening peak and fast pace in DD (8)(9)(10). Because PDF increases cAMP levels in at least some PDFR-expressing neurons (8,9,11,12), enhanced cAMP levels may slow the intrinsically fast pace of some PDFRcontaining clock neurons. cAMP may then affect clock speed only indirectly or by directly impacting the clock machinery.…”

Light-arousal and circadian photoreception circuits intersect at the large PDF cells of the Drosophila brain

“…6 B and C) and a broader circuit view of circadian entrainment: The l-LNvs are directly photosensitive in the late night and signal through PDF receptors. Although the precise expression pattern of PDFR in different neurons remains unknown, many clock neurons show responses to PDF (27). Therefore, the light information perceived by l-LNvs may be transmitted to the clock network by PDF and through routes that may even include some nonclock cells.…”