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

Wu Y, Cao G, Pavlicek B, Luo X, Nitabach MN - PLoS Biol. (2008)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Cellular and Molecular Physiology, Yale School of Medicine, New Haven, CT, USA.

ABSTRACT
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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Membrane-Tethered δ-ACTX-Hv1a Expression in LNV Neurons Induces Short Period of PDP1 Oscillation in LNDs in DDRed anti-PDP1 immunofluorescence (A) reveals PDP1 accumulation in LND nuclei. Bar graphs (B) show the number of neurons labeled with anti-PDP1 immunofluorescence (mean ± SEM). Control pdf>μO-MrVIA flies exhibit a similar temporal pattern of PDP1 accumulation in the LNDs as in the sLNVs, with peak centered around CT22 (p < 0.001). Experimental pdf>δ-ACTX-Hv1a flies exhibit similar temporal pattern of PDP1 oscillation to control pdf>μO-MrVIA flies on DD-D2 with peak at CT22 and trough at CT10. On DD-D4, LNDs of pdf>δ-ACTX-Hv1a flies exhibit damped peak of PDP1 oscillation at CT22 and trough at CT2-CT6 (p < 0.001). However, by DD-D6, a phase advance of PDP1 accumulation in the nuclei of LNDs induced by δ-ACTX-Hv1a expression manifests, with a peak at CT10 and trough at CT22-CT2 (p < 0.001). δ-ACTX-Hv1a expression in LNVs does not affect PDP1 oscillation in LND neurons in LD. Differences among different genotypes at different circadian times were compared using ANOVA with Tukey-Kramer multiple comparisons. n > 22 brain hemispheres for each experimental group, and error bars indicate SEM.
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pbio-0060273-g010: Membrane-Tethered δ-ACTX-Hv1a Expression in LNV Neurons Induces Short Period of PDP1 Oscillation in LNDs in DDRed anti-PDP1 immunofluorescence (A) reveals PDP1 accumulation in LND nuclei. Bar graphs (B) show the number of neurons labeled with anti-PDP1 immunofluorescence (mean ± SEM). Control pdf>μO-MrVIA flies exhibit a similar temporal pattern of PDP1 accumulation in the LNDs as in the sLNVs, with peak centered around CT22 (p < 0.001). Experimental pdf>δ-ACTX-Hv1a flies exhibit similar temporal pattern of PDP1 oscillation to control pdf>μO-MrVIA flies on DD-D2 with peak at CT22 and trough at CT10. On DD-D4, LNDs of pdf>δ-ACTX-Hv1a flies exhibit damped peak of PDP1 oscillation at CT22 and trough at CT2-CT6 (p < 0.001). However, by DD-D6, a phase advance of PDP1 accumulation in the nuclei of LNDs induced by δ-ACTX-Hv1a expression manifests, with a peak at CT10 and trough at CT22-CT2 (p < 0.001). δ-ACTX-Hv1a expression in LNVs does not affect PDP1 oscillation in LND neurons in LD. Differences among different genotypes at different circadian times were compared using ANOVA with Tukey-Kramer multiple comparisons. n > 22 brain hemispheres for each experimental group, and error bars indicate SEM.

Mentions: We used anti-PDP1 immunofluorescence to assess the effects of phase-advanced PDF cycling in δ-ACTX-Hv1a–expressing PDF-secreting LNVs on cellular oscillation of non-LNV clock neurons that express functional PDF receptor [26]. Control pdf>μO-MrVIA flies exhibit a similar temporal pattern of PDP1 accumulation in the LND cell group in both LD and DD conditions, with peak levels late at night or subjective night, and trough levels during day or subjective day (Figure 10; p < 0.001). This constant phase of PDP1 accumulation over 6 d in DD indicates that LND cellular oscillation free-runs with approximately 24-h period in control flies. Experimental pdf>δ-ACTX-Hv1a flies exhibit a similar temporal pattern of LND PDP1 oscillation to control pdf>μO-MrVIA flies in LD and on DD-D2. However, by DD-D4, PDP1 oscillation in LNDs of experimental pdf>δ-ACTX-Hv1a flies has begun to phase shift relative to control, with blunting of peak accumulation late in subjective night (p < 0.001). By DD-D6, the peak of LND PDP1 accumulation in flies expressing membrane-tethered δ-ACTX-Hv1a in LNVs has phase advanced to CT10. This phase advance indicates that membrane-tethered δ-ACTX-Hv1a expression in the LNVs induces an increase in the pace of LND cellular oscillation. The approximately 12-h phase advance that accumulates after 6 d in DD is consistent with the approximately 22-h free-running period of the short-period component of the complex locomotor rhythms exhibited by many pdf>δ-ACTX-Hv1a flies.


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

Wu Y, Cao G, Pavlicek B, Luo X, Nitabach MN - PLoS Biol. (2008)

Membrane-Tethered δ-ACTX-Hv1a Expression in LNV Neurons Induces Short Period of PDP1 Oscillation in LNDs in DDRed anti-PDP1 immunofluorescence (A) reveals PDP1 accumulation in LND nuclei. Bar graphs (B) show the number of neurons labeled with anti-PDP1 immunofluorescence (mean ± SEM). Control pdf>μO-MrVIA flies exhibit a similar temporal pattern of PDP1 accumulation in the LNDs as in the sLNVs, with peak centered around CT22 (p < 0.001). Experimental pdf>δ-ACTX-Hv1a flies exhibit similar temporal pattern of PDP1 oscillation to control pdf>μO-MrVIA flies on DD-D2 with peak at CT22 and trough at CT10. On DD-D4, LNDs of pdf>δ-ACTX-Hv1a flies exhibit damped peak of PDP1 oscillation at CT22 and trough at CT2-CT6 (p < 0.001). However, by DD-D6, a phase advance of PDP1 accumulation in the nuclei of LNDs induced by δ-ACTX-Hv1a expression manifests, with a peak at CT10 and trough at CT22-CT2 (p < 0.001). δ-ACTX-Hv1a expression in LNVs does not affect PDP1 oscillation in LND neurons in LD. Differences among different genotypes at different circadian times were compared using ANOVA with Tukey-Kramer multiple comparisons. n > 22 brain hemispheres for each experimental group, and error bars indicate SEM.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2577701&req=5

pbio-0060273-g010: Membrane-Tethered δ-ACTX-Hv1a Expression in LNV Neurons Induces Short Period of PDP1 Oscillation in LNDs in DDRed anti-PDP1 immunofluorescence (A) reveals PDP1 accumulation in LND nuclei. Bar graphs (B) show the number of neurons labeled with anti-PDP1 immunofluorescence (mean ± SEM). Control pdf>μO-MrVIA flies exhibit a similar temporal pattern of PDP1 accumulation in the LNDs as in the sLNVs, with peak centered around CT22 (p < 0.001). Experimental pdf>δ-ACTX-Hv1a flies exhibit similar temporal pattern of PDP1 oscillation to control pdf>μO-MrVIA flies on DD-D2 with peak at CT22 and trough at CT10. On DD-D4, LNDs of pdf>δ-ACTX-Hv1a flies exhibit damped peak of PDP1 oscillation at CT22 and trough at CT2-CT6 (p < 0.001). However, by DD-D6, a phase advance of PDP1 accumulation in the nuclei of LNDs induced by δ-ACTX-Hv1a expression manifests, with a peak at CT10 and trough at CT22-CT2 (p < 0.001). δ-ACTX-Hv1a expression in LNVs does not affect PDP1 oscillation in LND neurons in LD. Differences among different genotypes at different circadian times were compared using ANOVA with Tukey-Kramer multiple comparisons. n > 22 brain hemispheres for each experimental group, and error bars indicate SEM.
Mentions: We used anti-PDP1 immunofluorescence to assess the effects of phase-advanced PDF cycling in δ-ACTX-Hv1a–expressing PDF-secreting LNVs on cellular oscillation of non-LNV clock neurons that express functional PDF receptor [26]. Control pdf>μO-MrVIA flies exhibit a similar temporal pattern of PDP1 accumulation in the LND cell group in both LD and DD conditions, with peak levels late at night or subjective night, and trough levels during day or subjective day (Figure 10; p < 0.001). This constant phase of PDP1 accumulation over 6 d in DD indicates that LND cellular oscillation free-runs with approximately 24-h period in control flies. Experimental pdf>δ-ACTX-Hv1a flies exhibit a similar temporal pattern of LND PDP1 oscillation to control pdf>μO-MrVIA flies in LD and on DD-D2. However, by DD-D4, PDP1 oscillation in LNDs of experimental pdf>δ-ACTX-Hv1a flies has begun to phase shift relative to control, with blunting of peak accumulation late in subjective night (p < 0.001). By DD-D6, the peak of LND PDP1 accumulation in flies expressing membrane-tethered δ-ACTX-Hv1a in LNVs has phase advanced to CT10. This phase advance indicates that membrane-tethered δ-ACTX-Hv1a expression in the LNVs induces an increase in the pace of LND cellular oscillation. The approximately 12-h phase advance that accumulates after 6 d in DD is consistent with the approximately 22-h free-running period of the short-period component of the complex locomotor rhythms exhibited by many pdf>δ-ACTX-Hv1a flies.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Cellular and Molecular Physiology, Yale School of Medicine, New Haven, CT, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus