Drosophila clock neurons are self-sustaining cellular oscillators that rely on negative transcriptional feedback to keep circadian time. Proper regulation of organismal rhythms of physiology and behavior requires coordination of the oscillations of individual clock neurons within the circadian control network. Over the last decade, it has become clear that a key mechanism for intercellular communication in the circadian network is signaling between a subset of clock neurons that secrete the neuropeptide pigment dispersing factor (PDF) and clock neurons that possess its G protein-coupled receptor (PDFR). Furthermore, the specific hypothesis has been proposed that PDF-secreting clock neurons entrain the phase of organismal rhythms, and the cellular oscillations of other clock neurons, via the temporal patterning of secreted PDF signals. In order to test this hypothesis, we have devised a novel technique for altering the phase relationship between circadian transcriptional feedback oscillation and PDF secretion by using an ion channel–directed spider toxin to modify voltage-gated Na + channel inactivation in vivo. This technique relies on the previously reported “tethered-toxin” technology for cell-autonomous modulation of ionic conductances via heterologous expression of subtype-specific peptide ion channel toxins as chimeric fusion proteins tethered to the plasma membrane with a glycosylphosphatidylinositol (GPI) anchor. We demonstrate for the first time, to our knowledge, the utility of the tethered-toxin technology in a transgenic animal, validating four different tethered spider toxin ion channel modifiers for use in Drosophila. Focusing on one of these toxins, we show that GPI-tethered Australian funnel-web spider toxin δ-ACTX-Hv1a inhibits Drosophila para voltage-gated Na + channel inactivation when coexpressed in Xenopus oocytes. Transgenic expression of membrane-tethered δ-ACTX-Hv1a in vivo in the PDF-secreting subset of clock neurons induces rhythmic action potential bursts and depolarized plateau potentials. These in vitro and in vivo electrophysiological effects of membrane-tethered δ-ACTX-Hv1a are consistent with the effects of soluble δ-ACTX-Hv1a purified from venom on Na + channel physiological and biophysical properties in cockroach neurons. Membrane-tethered δ-ACTX-Hv1a expression in the PDF-secreting subset of clock neurons induces an approximately 4-h phase advance of the rhythm of PDF accumulation in their terminals relative to both the phase of the day:night cycle and the phase of the circadian transcriptional feedback loops. As a consequence, the morning anticipatory peak of locomotor activity preceding dawn, which has been shown to be driven by the clocks of the PDF-secreting subset of clock neurons, phase advances coordinately with the phase of the PDF rhythm of the PDF-secreting clock neurons, rather than maintaining its phase relationship with the day:night cycle and circadian transcriptional feedback loops. These results (1) validate the tethered-toxin technology for cell-autonomous modulation of ion channel biophysical properties in vivo in transgenic Drosophila, (2) demonstrate that the kinetics of para Na + channel inactivation is a key parameter for determining the phase relationship between circadian transcriptional feedback oscillation and PDF secretion, and (3) provide experimental support for the hypothesis that PDF-secreting clock neurons entrain the phase of organismal rhythms via the temporal patterning of secreted PDF signals.
The regulation of the daily fluctuations that characterize an organism's physiology and behavior requires coordination of the cellular oscillations of individual “clock” neurons within the circadian control network. Clock neurons that secrete a neuropeptide called pigment dispersing factor (PDF) calibrate, or entrain, both the phase of organismal rhythms and the cellular oscillations of other clock neurons. In this study, we tested the hypothesis that phase of PDF secretion rhythms entrains phase of non-PDF neurons and locomotor rhythms using the tethered- toxin technique (which affixes toxins to the cell membrane) to express ion channel–specific peptide toxins in PDF neurons. A particular toxin inhibits inactivation of the Drosophila para sodium (Na +) channel. Inhibition of Na + channel inactivation in PDF neurons of transgenic flies induces phase advance of PDF rhythm, and correlated phase advance of lights-on anticipatory locomotor activity, suggesting that phase of morning activity is determined by phase of PDF oscillation. Therefore, voltage-gated Na + channels of Drosophila clock neurons play a key role in determining the phase relationship between circadian transcriptional feedback oscillation and PDF secretion, and PDF-secreting clock neurons entrain the phase of organismal rhythms via the temporal patterning of secreted PDF signals.
Cell-autonomous inhibition of Drosophila para Na + channel inactivation using a membrane-tethered spider toxin phase shifts circadian neuropeptide output from cellular oscillation, and the phase of morning anticipatory activity is determined by this phase-shifted neuropeptide output.