PLoS Biol 2008 Nov 04;611:e273. doi: 10.1371/journal.pbio.0060273.

Phase coupling of a circadian neuropeptide with rest/activity rhythms detected using a membrane-tethered spider toxin.

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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 delta-ACTX-Hv1a inhibits Drosophila para voltage-gated Na(+) channel inactivation when coexpressed in Xenopus oocytes. Transgenic expression of membrane-tethered delta-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 delta-ACTX-Hv1a are consistent with the effects of soluble delta-ACTX-Hv1a purified from venom on Na(+) channel physiological and biophysical properties in cockroach neurons. Membrane-tethered delta-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.

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Species referenced: Xenopus
Genes referenced: clock gnpda1 gpi lgals4.2 myc pdf pdp1 tub


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